Curr Opin Chem Biol, 2003 Feb, 7(1), 39 - 43 Protein expression systems for structural genomics and proteomics; Yokoyama S; One of the key steps of structural genomics and proteomics is high-throughput expression of many target proteins . Gene cloning, especially by ligation-independent cloning techniques, and recombinant protein expression using microbial hosts such as Escherichia coli and the yeast Pichia pastoris are well optimized and further robotized . Cell-free protein synthesis systems have been developed for large-scale production of protein samples for NMR (stable-isotope labeling) and X-ray crystallography (selenomethionine substitution) . Protein folding is still a major bottleneck in protein expression . Cell-based and cell-free methods for screening of suitable samples for structure determination have been developed for achieving a high success rate. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 154 - 60 {Influence of signal peptide sequences on the expression of heterogeneous proteins in Pichia pastoris}; Xiong AS et al.; Pichia pastoris has been developed to be a very efficient expression host for the heterogeneous proteins since its alcohol promoter was isolated and cloned, and its transformation technique was established . For further improving the secretion expression of heterogeneous proteins, in this research, the signal sequences were studied . At first, the Saccharomyces cerevisiae mating factor alpha prepro-leader sequence was synthesized using successive PCR and designated as MF4I . Then, ten different signal sequences were constructed by adding the N-terminal residues of Pichia pastoris Aox1 protein to the N-terminal of the MF4I . These ten signal sequences were used for directing phytase gene secretion in Pichia pastoris, the secretion of phytase were increased in Pichia pastoris strains containing new signal sequence . Among these strains, the phytase secretion was highest in strain contain signal sequence added with A, I, P three Aox1 N-terminal residues; the phytase secretion of Pichia pastoris was 90 mg/L in flake . The secretion was six-fold of that with original Saccharomyces cerevisiae mating factor alpha prepro-leader sequence . In addition, insert of ten residues E E A E A E A E P K can further increase the phytase secretion by 35%, the secretion reach 120 mg/L. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 127 - 32 {Enhancement of the production of SAM by overexpression of SAM synthetase in Pichia pastoris}; Yu ZL et al.; S-Adenosyl-L-methionine(SAM) is an important metabolic intermediate in the metabolic flux of sulphur . SAM is involved in three key metabolic pathways: transmethylation, transsulfuration and polyamine synthesis . As a potential therapeutic agent, SAM is being used as over the counter drug and nutrient supplement . An expression vector, harboring SAM synthetase 2 gene from S . cerevisiae and regulated by the glyceraldehyde-3-phosphate dehydrogenase gene promoter P(GAP), was transformed into GS115 strain of P . pastoris . Through zeocin resistance and expression screening, a recombinant strain was obtained that had high SAM yield and the fermentation conditions were optimized . The results showed that carbon source, nitrogen source, pH and dissolved oxygen had significant effects on the accumulation of SAM . The SAM production of the recombinant cells reached 2.49 g/L after fermentation for three days under the optimized conditions . The present studies show that fermentation of recombinant P . pastoris strain, expressing heterologous SAM synthetase gene, may be a promising approach for the production of SAM. Acta Biochim Pol, 2002, 49(4), 959 - 68 The effect of vanadate on Pichia pastoris growth, protein kinase A activity and ribosomal protein phosphorylation; Frajnt M et al.; It was found that wild type yeast Pichia pastoris can tolerate vanadate concentration as high as 25 mM in the growth medium . Moreover, four vanadate-resistant P . pastoris strains designated JC100/1, JC100/3, JC100/9 and JC100/15 exhibiting tolerance up to 150 mM vanadate were selected . Growth of P . pastoris was correlated with vanadate to vanadyl reduction and its accumulation in the growth medium . In two selected strains, JC100/9 and JC100/15, protein kinase A activity was much higher in comparison to the wild type strain even without vanadate addition to the growth medium . Moreover, in the presence of vanadate, protein kinase A activity was significantly increased in the wild type and the vanadate-resistant JC100/1 and JC100/3 strains . It was also found that phosphorylation of a 40 kDa protein associated with ribosomes occured in all vanadate-resistant strains from the logarithmic, while in the wild type strain from the stationary growth phase . From the presented results it can be concluded that a protein kinase A signalling pathway(s) might be involved in the mechanism of P . pastoris vanadate resistance . The results also indicate a possible role of the 40 kDa protein in protection of P . pastoris against vanadate toxicity. Biochim Biophys Acta, 2003 Jan 31, 1645(1), 1 - 5 Change in maltose- and soluble starch-hydrolyzing activities of chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae; Sugimoto M et al.; The chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes . The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium . The two chimeric M . javanicus alpha-glucosidases, of which the N- and C-terminal regions are substituted for those of A . oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M . javanicus alpha-glucosidase, respectively . The chimeric enzymes changed on the V(max) values for maltose significantly, whereas the K(m) values were similar to that of the native enzyme. Ai Zheng, 2002 Nov, 21(11), 1197 - 202 {Inhibitory effects of recombinant human endostatin on growth and metastasis of lung adenocarcinoma LA795 in mice}; Xia H et al.; BACKGROUND & OBJECTIVE: Generally, growth and metastasis of tumor are critically dependent on angiogenesis . Endostatin can specifically inhibits tumor angiogenesis . The current study was designed to evaluate the inhibitory effect of recombinant human endostatin (rhES) secreted by pichia, pastoris, GS115 on the growth and metastasis of mice lung adenocarcinoma LA795 in mice T739 . METHODS: To select a strain that could highly express recombinant human endostatin, then induce the clone to express rhES by adding methanol . Purification of rhES was performed with heparin affinity chromatography . LA795 tumor cells were inoculated subcutaneously into the dorsa of T739 mice, and the mice were randomized into two groups . The first group was given rhES(20 mg/kg/d), and the second group was given equal volume of PBS, for 14 consecutive days . The volume of tumors were measured . And the tumor metastasis in the lungs of the mice was observed . RESULTS: The selected clone was induced to secrete enough soluble rhES . The purified protein could strongly inhibit growth and metastasis of mice lung adenocarcinoma LA795 in T739 mice (P < 0.001) . CONCLUSION: The rhES secreted by pichia, pastoris, GS115 has good biological activities and greatly inhibit growth and metastasis of mice lung adenocarcinoma LA795 in mice T739. J Biol Chem, 2003 Apr 18, 278(16), 14249 - 56 Epub 2003 Jan 13. Refined solution structure of a liganded type 2 wheat nonspecific lipid transfer protein; Pons JL et al.; The refined structure of a wheat type 2 nonspecific lipid transfer protein (ns-LTP2) liganded with l-alpha-palmitoylphosphatidylglycerol has been determined by NMR . The (15)N-labeled protein was produced in Pichia pastoris . Physicochemical conditions and ligandation were intensively screened to obtain the best NMR spectra quality . This ns-LTP2 is a 67-residue globular protein with a diameter of about 30 A . The structure is composed of five helices forming a right superhelix . The protein presents an inner cavity, which has been measured at 341 A(3) . All of the helices display hydrophobic side chains oriented toward the cavity . The phospholipid is found in this cavity . Its fatty acid chain is completely inserted in the protein, the l-alpha-palmitoylphosphatidylglycerol glycerol moiety being located on a positively charged pocket on the surface of the protein . The superhelix structure of the protein is coiled around the fatty acid chain . The overall structure shows similarities with ns-LTP1 . Nevertheless, large three-dimensional structural discrepancies are observed for the H3 and H4 alpha-helices, the C-terminal region, and the last turn of the H2 helix . The lipid is orthogonal to the orientation observed in ns-LTP1 . The volume of the hydrophobic cavity appears to be in the same range as the one of ns-LTP1, despite the fact that ns-LTP2 is shorter by 24 residues. Indian J Med Res, 2002 Jul, 116, 5 - 12 Change in distribution & antifungal susceptibility of Candida species isolated from candidaemia cases in a tertiary care centre during 1996-2000; Chakrabarti A et al.; BACKGROUND & OBJECTIVES: As a marked increase in the number of patients with candidaemia was reported in the first half (1991-1995) of the last decade at the Postgraduate Institute of Medical Education & Research, Chandigarh, India, the present study was aimed at determining further change if any, in the incidence and distribution of Candida species and their antifungal resistance pattern during the second half (1996-2000) of the same decade . METHODS: The patients with candidaemia were studied to determine the frequency of candidaemia and Candida species isolated during 1996-2000 . One hundred Candida strains other than Pichia anomala (C . pelliculosa) were randomly selected from those isolates to evaluate antifungal susceptibility pattern against amphotericin B, 5-fluorocytosine, ketoconazole, fluconazole and itraconazole . The results were compared with our previous study . RESULTS: An increase in the number of patients with candidaemia was observed during 1996 (538) and 1997 (421) compared to 1998-2000 due to P . anomala outbreak . With the control of the outbreak, a substantial decrease in the incidence of candidaemia was observed from 1998 (251 in 1998, 122 in 1999 and 165 in 2000) . A higher isolation of non-C . albicans Candida species (89.8%) was observed, with C . tropicalis being the most common (541, 36.1%) agent . No major change in the isolation rate of other non-C . albicans Candida species (C . guilliermondii, C . krusei, C . glabrata and C . parapsilosis) was observed . An emergence of resistance to amphotericin B in 15.4 per cent C . albicans, 8.1 per cent C . tropicalis and 33.3 per cent C . krusei strains was observed . An increase in resistance to ketoconazole (from 0% to 13%) and 5-fluorocytosine (from 1% to 8%) and a decrease to fluconazole (from 13% to 6%) were observed . Resistance to itraconazole was observed in 17 per cent of Candida strains by broth macro-dilution method . INTERPRETATION & CONCLUSION: A change in the isolation of Candida species was observed i.e . in the incidence and isolation of non-C . albicans Candida species . Emergence of resistance to amphotericin B and increase of resistance to most other antifungals are cause for concern. J Biol Chem, 2003 Mar 7, 278(10), 8257 - 60 Epub 2003 Jan 03. Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase . Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms; Rozanov DV et al.; An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies . A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces . The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway . To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris . Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography . Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis . This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites . Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112) . The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)) . These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation . Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells. Appl Environ Microbiol, 2003 Jan, 69(1), 495 - 503 Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity; Jin YS et al.; D-Xylulokinase (XK) is essential for the metabolism of D-xylose in yeasts . However, overexpression of genes for XK, such as the Pichia stipitis XYL3 gene and the Saccharomyces cerevisiae XKS gene, can inhibit growth of S . cerevisiae on xylose . We varied the copy number and promoter strength of XYL3 or XKS1 to see how XK activity can affect xylose metabolism in S . cerevisiae . The S . cerevisiae genetic background included single integrated copies of P . stipitis XYL1 and XYL2 driven by the S . cerevisiae TDH1 promoter . Multicopy and single-copy constructs with either XYL3 or XKS1, likewise under control of the TDH1 promoter, or with the native P . stipitis promoter were introduced into the recombinant S . cerevisiae . In vitro enzymatic activity of XK increased with copy number and promoter strength . Overexpression of XYL3 and XKS1 inhibited growth on xylose but did not affect growth on glucose even though XK activities were three times higher in glucose-grown cells . Growth inhibition increased and ethanol yields from xylose decreased with increasing XK activity . Uncontrolled XK expression in recombinant S . cerevisiae is inhibitory in a manner analogous to the substrate-accelerated cell death observed with an S . cerevisiae tps1 mutant during glucose metabolism . To bypass this effect, we transformed cells with a tunable expression vector containing XYL3 under the control of its native promoter into the FPL-YS1020 strain and screened the transformants for growth on, and ethanol production from, xylose . The selected transformant had approximately four copies of XYL3 per haploid genome and had moderate XK activity . It converted xylose into ethanol efficiently. Clin Chem, 2003 Jan, 49(1), 77 - 86 Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay; Kapadia C et al.; BACKGROUND: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids . METHODS: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system . The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies . A sandwich-type immunoassay was developed with these antibodies . The assay was used to measure hK13 in various biological fluids and tissue extracts . Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections . RESULTS: The hK13 immunoassay had a detection limit of 0.05 micro g/L and showed no cross-reactivity with homologous kallikreins . The assay was linear at 0-20 micro g/L, and within-and between-run CVs were <10% (n = 12) . hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate . hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues . hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate . hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues . Recovery of active enzyme added to milk or amniotic fluid was 70-98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors . In fluids and tissue extracts, hK13 was found in its free (approximately 30 kDa) form . CONCLUSIONS: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease. Biosci Biotechnol Biochem, 2002 Nov, 66(11), 2297 - 305 Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening; Kawakami A et al.; We isolated two cDNAs of winter wheat (Triticum aestivum L.), designated wft1 and wft2, which encoded sucrose:fructan 6-fructosyltransferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), respectively, which are involved in the synthesis of fructan in wheat . wft1 and wft2 were cloned by screening of a cDNA library with probed-cDNA fragments corresponding to plant fructosyltransferase and invertase . The identity of the clones was verified by functional characterization of recombinant proteins expressed in methylotrophic yeast, Pichiapastoris . Northern blotting showed that the level of wft2 transcripts increased from autumn to early winter in the crown tissues of all field-grown wheat cultivars examined . Higher levels of wft1 and wft2 transcripts were found in leaf tissues of snow mold-resistant cultivars, which accumulated more fructan than other cultivars . Our results showed that Wft1 and Wft2 were important in fructan accumulation during cold hardening of winter wheat. J Gen Appl Microbiol, 1999 Oct, 45(5), 239 - 246 A phylogenetic study of ubiquinone Q-8 species of the genera Candida, Pichia, and Citeromyces based on 18S ribosomal DNA sequence divergence; Suzuki M et al.; To clarify phylogenetic relationships among species of the anamorphic ascomycetous genus Candida with ubiquinone Q-8, we determined complete sequences of 18S ribosomal RNA genes (18S rDNAs) from the type strains of 20 species of the genus Candida and 7 of the teleomorphic ascomycetous genera Pichia and Citeromyces, which have Q-8 as the major ubiquinone . Q-8-forming Candida species were divided into six clusters and were phylogenetically distant from a group of Candida species that included the type species of the genus . One Q-8-forming species from each of the genera Pichia, Citeromyces, or Clavispora was included in five of six clusters . Cluster 1 comprised C . ishiwadae, C . ernobii, C . karawaiewii, C . anatomiae, C . populi, and Pichia holstii . Cluster 2 comprised C . globosa and its teleomorph, Citeromyces matritensis . Cluster 3 comprised C . molischiana and Pichia capsulata . Cluster 4 comprised C . silvanorum, C . sequanensis, C . fennica, C . entomophila, C . homilentoma, C . rhagii, C . gotoi, and Pichia burtonii . Cluster 5 comprised C . fructus, C . musae, and C . lusitaniae (anamorph of Clavispora lusitaniae) . Cluster 6 comprised C . stellata, C . lactiscondensi, C . galacta, and C . incommunis and was a heterogeneous group with large interspecific divergence . Pichia pastoris was quite divergent and phylogenetically distant from other Pichia species examined . Pichia methanolica and its synonym, P . cellobiosa, which have both Q-7 and Q-8 as major ubiquinones, were closely associated with Q-7-forming Williopsis salicorniae . Based on this comparative analysis of 18S rDNA sequences, it is evident that Q-8 Candida species and Q-8 Pichia species are polyphyletic. J Gen Appl Microbiol, 1999 Oct, 45(5), 229 - 238 A phylogenetic study on galactose-containing Candida species based on 18S ribosomal DNA sequences; Suzuki M et al.; Phylogenetic relationships of 33 Candida species containing galactose in the cells were investigated by using 18S ribosomal DNA sequence analysis . Galactose-containing Candida species and galactose-containing species from nine ascomycetous genera were a heterogeneous assemblage . They were divided into three clusters (II, III, and IV) which were phylogenetically distant from cluster I, comprising 9 galactose-lacking Candida species, C . glabrata, C . holmii, C . krusei, C . tropicalis (the type species of Candida), C . albicans, C . viswanathii, C . maltosa, C . parapsilosis, C . guilliermondii, and C . lusitaniae, and 17 related ascomycetous yeasts . These three clusters were also phylogenetically distant from Schizosaccharomyces pombe, which contains galactomannan in its cell wall . Cluster II comprised C . magnoliae, C . vaccinii, C . apis, C . gropengiesseri, C . etchellsii, C . floricola, C . lactiscondensi, Wickerhamiella domercqiae, C . versatilis, C . azyma, C . vanderwaltii, C . pararugosa, C . sorbophila, C . spandovensis, C . galacta, C . ingens, C . incommunis, Yarrowia lipolytica, Galactomyces geotrichum, and Dipodascus albidus . Cluster III comprised C . tepae, C . antillancae and its synonym C . bondarzewiae, C . ancudensis, C . petrohuensis, C . santjacobensis, C . ciferrii (anamorph of Stephanoascus ciferrii), Arxula terrestris, C . castrensis, C . valdiviana, C . paludigena, C . blankii, C . salmanticensis, C . auringiensis, C . bertae, and its synonym C . bertae var . chiloensis, C . edax (anamorph of Stephanoascus smithiae), Arxula adeninivorans, and C . steatolytica (synonym of Zygoascus hellenicus) . Cluster IV comprised C . cantarellii, C . vinaria, Dipodascopsis uninucleata, and Lipomyces lipofer . Two galactose-lacking and Q-8-forming species, C . stellata and Pichia pastoris, and 5 galactose-lacking and Q-9-forming species, C . apicola, C . bombi, C . bombicola, C . geochares, and C . insectalens, were included in Cluster II . Two galactose-lacking and Q-9-forming species, C . drimydis and C . chiropterorum, were included in Cluster III. Genetika, 2002 Nov, 38(11), 1451 - 62 {Formation of ARS-independent miniplasmids upon transformation of yeast Pichia methanolica with DNA molecules containing "transforming" and "nontransforming" genes}; Tarutina MG et al.; In the study of transforming DNA molecules in cells of the methylotrophic yeast Pichia methanolica, it was found that the efficiency of transformation and the possibility of autonomous replication of hybrid plasmids and miniplasmids consisting of only transforming gene (Trg) sequences do not depend on the presence of ARS sequences with the canonical structure, such as 2 microns DNA in the yeast Saccharomyces cerevisiae . Data supported a suggestion about the existence of the relationship between gene transforming activity, its replicative ability, and transcription . The efficiency of vector activity of Trg is not sufficient to provide the transformation of other sequences of transforming DNA (tDNA) . The loss of nonselected sequences of tDNA observed upon transformation is the result of rearrangements and deletions and is associated with the activity of cellular systems involved in nucleic acid metabolism . Miniplasmids similar in structure were shown to differ in their stability in independent transformants . The rate of loss of autonomously replicating plasmids strongly varied (from 1 to 99%) under nonselective conditions and nearly did not depend on plasmid structure. Zhonghua Nan Ke Xue, 2002, 8(4), 292 - 4, 298 {Advances in eukaryotic expression systems}; Gao Y et al.; The increasing popularity of eukaryotic expression systems can be attributed to their capability of performing many post-translational modifications . At present, There mainly are three expression systems including yeast expression system, insect cell expression system and mammalian cell expression system . The methylotropic yeast Pichia Pastoris usually utilizes alcohol oxidase promoter to drive the expression of foreign gene . Recently, a continuous fermentation has been developed in Pichia Pastoris with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter . The baculovirus-mediated insect cell expression system is considered to be safe, powerful, but cell-lytic . Baculovirus-S2 system uses the popular and genetically well understood Drosphila S2 cells which do not appear to be lysed after infection . In mammalian cell expression system, recombinant adenovirus are attracting a great deal of attention as a highly efficient gene transfer vehicle . The frequency of Ad vector rescue by homologous recombination in E . coli and Cre-mediated site-specific recombination is significantly higher than by homologous recombination in vivo . Tetracycline-regulatable system is a widely used mammalian cell inducible expression system due to its high efficiency and stringency. Yeast, 2003 Jan 15, 20(1), 39 - 51 Analysis of the hypoxia-induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast; Passoth V et al.; We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP . This system was used to analyse hypoxia-dependent PsADH2 regulation . Reporter gene activity was only found under oxygen limitation on a fermentable carbon source . The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae . Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction . This motif was different from the known hypoxia response elements of S . cerevisiae but showed some similarity to the mammalian HIF-1 binding site . Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation . Similar to the S . cerevisiae heme sensor system, the promoter was induced by Co(2+) . Cyanide was not able to mimic the effect of oxygen limitation . The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co(2+) but not by cyanide . Thus, the very first promoter analysis in P . stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast . Biochemistry, 2002 Dec 24, 41(51), 15195 - 202 Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography; Kawano S et al.; Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid . We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography . The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model . The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds . The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89 . In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79 . Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid. Clin Chim Acta, 2003 Jan, 327(1-2), 171 - 9 Expression of TIMP-1 in Pichia pastoris . Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library; Wozniak G et al.; To quantitate tissue inhibitor of metalloproteinase (TIMP)-1 in biological samples, a strategy for isolation of monoclonal antibodies was applied that employs a phage-displayed single-chain Fv (scFv) . In order to obtain sufficient amounts of TIMP-1 to use as an antigen, high-level expression in Pichia pastoris was achieved under the control of the AOX-1 promotor . Purified protein antigen was then used for panning to achieve enrichment of specific phage from naive scFv library . In five subsequent panning rounds, antibody fragments that display specificity to TIMP-1 were selected . Regions encoding scFv were subcloned into a vector allowing production of scFv-alkaline phosphatase (AP) fusion proteins . Two such conjugates displaying dose-dependent reactivity with TIMP-1 were isolated and characterised, providing the basis for the construction of a TIMP-1 quantitation assay based entirely on recombinant proteins. Microbiology, 2002 Dec, 148(Pt 12), 4003 - 14 Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host; Soden DM et al.; The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter . The native Ple . sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic . pastoris . The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization . The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1 . The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38 . The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively . With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5 . The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 115 - 24 Enhancement of proteinase inhibitory activity of recombinant human cystatin C using random-centroid optimization; Ogawa M et al.; We had previously written a random-centroid optimization computer program for genetics (RCG) to optimize protein engineering, which was successfully applied to modify single site of the 16 amino acid residues at the active site of B . stearothermophilys neutral protease for improving thermostability {J . Agric . Food Chem., 46 (1998) 1655} . The same program was applied in this study to double-site mutation of the entire sequence of human cystatin C (HCC) with 120 residues for improving its protease inhibitory activity . The RCG program selected two sites simultaneously and amino acid residues to replace the sites selected in the sequence in order to find the best papain-inhibitory activity and stability of the protease inhibitor . Twenty-three double mutants and twenty-two single mutants were expressed by Pichia pastoris . Of the total 45 mutants, G12W/H86V mutant showed a 5-fold increase in the bioactivity over the recombinant wild-type (WT) cystatin . Also, P13F mutant exhibited a half-life temperature (T1/2) 5.2 degrees C higher than 68.2 degrees C of WT in addition to a 56% greater papain inhibitory activity . Mutation for diminishing beta-sheet content reduced polymerization of cystatin C, thus improving papain-inhibitory activity . The approach using RCG was able to improve the functional properties of cystatin by least relying on the prior knowledge of its molecular structure. Curr Genet, 2002 Nov, 42(2), 114 - 22 Epub 2002 Nov 21. Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T . harzianum T-203); Viterbo A et al.; The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups . CHIT37 from T . harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level . Northern analysis showed that chit36Y from T . asperellum is regulated both by glucose and nitrogen levels . Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene . The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants . Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression . The R . solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (<12 kDa) . CHIT36 recombinant protein from the yeast Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains. Biotechnol Bioeng, 2003 Feb 5, 81(3), 291 - 8 Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica; Mayson BE et al.; The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter . Methanol concentrations during the induction phase directly affect cellular growth and protein yield . Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P . methanolica expressing the human transferrin N-lobe protein . The PMAD18 P . methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype . Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration . Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time . J Gen Appl Microbiol, 2002 Feb, 48(1), 55 - 65 A phylogenetic study of ubiquinone-7 species of the genus Candida based on 18S ribosomal DNA sequence divergence; Suzuki M et al.; To clarify phylogenetic relationships among ubiquinone 7 (Q7)-forming species of the genus Candida, we analyzed the nearly complete sequences of 18S ribosomal RNA genes (18S rDNAs) from fifty strains (including 46 type strains) of Candida species, and from 8 type strains of species/varieties of the genera Issatchenkia, Pichia and Saturnispora . Q7-forming Candida species were divided into three major groups (Group I, II, and III) and were phylogenetically distant from a group that includes the type species of the genus Candida . Group I included four clusters with basal branches that were weakly supported . The first cluster comprised C . vartiovaarae, C . maritima, C . utilis, C . freyschussii, C . odintsovae, C . melinii, C . quercuum, Williopsis saturnus var . saturnus, and W . mucosa . The second cluster comprised C . norvegica, C . montana, C . stellimalicola, C . solani, C . berthetii, and C . dendrica . Williopsis pratensis, W . californica, Pichia opuntiae and 2 related species, P . amethionina (two varieties), and P . caribaea were also included in this cluster . The third cluster comprised C . pelliculosa (anamorph of P . anomala), C . nitrativorans, and C . silvicultrix . The fourth cluster comprised C . wickerhamii and C . peltata, which were placed in the P . holstii - C . ernobii clade with Q8-containing species . Group II comprised C . pignaliae, C . nemodendra, C . methanolovescens, C . maris, C . sonorensis, C . pini, C . llanquihuensis, C . cariosilignicola, C . ovalis, C . succiphila (including its two synonyms), C . methanosorbosa, C . nitratophila, C . nanaspora, C . boidinii (including its two synonyms), W . salicorniae, and P . methanolica . Group III was composed of four clusters with strong bootstrap support . The first cluster comprised C . valida (anamorph of P . membranifaciens), C . ethanolica, C . pseudolambica, C . citrea, C . inconspicua, C . norvegensis, C . rugopelliculosa, and C . lambica . Three species and two varieties of the genus Issatchenkia were also included in this cluster . The second cluster comprised C . diversa, C . silvae, 4 Saturnispora species, and P . besseyi . The third comprised C . sorboxylosa, and the fourth comprised C . vini . Based on this 18S rDNA sequence analysis, it is evident that Q7-forming Candida species and the genera Pichia and Williopsis are polyphyletic . The genus Issatchenkia is suggested to be congeneric with the genus Pichia . The genus Saturnispora is phylogenetically definable. Biotechnol Prog, 2002 Nov-Dec, 18(6), 1392 - 9 Design of methanol Feed control in Pichia pastoris fermentations based upon a growth model; Zhang W et al.; The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut(+) (methanol utilization plus) strain . Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity . An accurate methanol control is required in the methanol fed-batch phase (induction phase) in the fermentation . A simple "on-off" control strategy is inadequate for precise control of methanol concentrations in the fermentor . In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model . The closed-loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor . First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters K(c) (controller gain), tau(I) (controller integral time constant), and tau(D) (controller derivative time constant) . Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed-batch phase . Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings . The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved . The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes. Reprod Fertil Dev, 2002, 14(5-6), 327 - 32 Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris; Fidler AE et al.; The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians . To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris . The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCE The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro . Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF . This source of biologically active marsupial SCF may prove useful in future studies of marsupial development. Protein Expr Purif, 2002 Dec, 26(3), 496 - 505 Expression of CB2 cannabinoid receptor in Pichia pastoris; Feng W et al.; To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris . The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P . pastoris alcohol oxidase 1 gene . A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification . In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins . Radioligand binding assays demonstrated that the CB2 receptors expressed in P . pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems . Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin . MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor . ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor . In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P . pastoris for purification. Protein Expr Purif, 2002 Dec, 26(3), 406 - 15 Expression and characterization of a synthetic protein C activator in Pichia pastoris; Kunes YZ et al.; Protein C activators are proteases that activate protein C in the mammalian coagulation system . A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway . We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence . This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence . A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth . A two-step purification scheme was devised to purify ILPCA to approximately 80% purity . The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa . Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1) . In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations. Protein Expr Purif, 2002 Dec, 26(3), 394 - 405 Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris; Baumgartner P et al.; The kidney bean lectin Phaseolus vulgaris phytohemagglutinin E-form (PHA-E) was expressed and secreted by the methylotrophic yeast Pichia pastoris . To optimise yields of PHA-E, transformants of P . pastoris were selected for high-level production of the recombinant protein . A scaleable process for the production and purification of gram quantities of recombinant PHA-E is reported . PHA-E was secreted at approximately 100 mg/L at the 2- and 200-L scale and was purified to 95% homogeneity in a single step using cation-exchange chromatography . The purified recombinant PHA-E consists of four forms with molecular masses between 28.5 and 31.5 kDa, as assessed by MALDI-TOF, whereas its native counterpart has a molecular mass of approximately 30.5 kDa . Endoglycosidase treatment revealed that the range in size of the recombinant protein was attributed to differences in the nature of the N-linked oligosaccharides bound to the protein . The primary amino acid sequence of the recombinant PHA-E was found to be identical to the native protein and to have an agglutination activity similar to that of native PHA-E . The data presented here suggest that, using P . pastoris, gram quantities of a recombinant phytohemagglutinin E-form can be produced and that the recombinant protein is similar to the protein synthesised in plants with respect to structure and biological activity. Protein Expr Purif, 2002 Dec, 26(3), 386 - 93 Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent; Brucato CL et al.; Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains . It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation . The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies . To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris . Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity . A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility. Protein Expr Purif, 2002 Dec, 26(3), 349 - 56 Purification of full-length recombinant human and rat type 1 11beta-hydroxysteroid dehydrogenases with retained oxidoreductase activities; Nobel CS et al.; 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum . This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase . Previous attempts to purify the native enzyme have yielded a protein without reductase activity . To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11beta-HSD1 with retention of their natural oxidoreductase activities . This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography . Both enzymes were 90-95% pure and exhibited dehydrogenase and reductase activities with K(M) values in agreement with those reported in the literature. J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 25, 782(1-2), 307 - 16 Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions; Weinberger SR et al.; Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed . When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species . The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations . In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution . Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS) . The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces . Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions . The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures . Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode . Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method. Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2177 - 9 Epub 2002 Nov 23. Crystallization of Pichia pastoris lysyl oxidase; Lee M et al.; A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms . PPLO is a glycoprotein . The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa) . Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution . Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution . Native data have been recorded from each crystal form at 100 K using synchrotron radiation . A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit. Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 241 - 6 Heterologous expression of cry2 gene from a local strain of Bacillus thuringiensis isolated in Nigeria; Ogunjimi AA et al.; A cry2 gene encoding a larvicidal crystal protein was isolated from a strain of Bacillus thuringiensis found in soil samples in Nigeria . This gene was cloned into plasmid pUC19 and subcloned into both pBluescript (sk(+/-)) and pPICZ alpha B placed under a T7/AOXI (alcohol oxidase I) promoter respectively and transformed into Escherichia coli and Pichia pastoris . Clones were induced for expression, and the cellular proteins extracted and analysed by SDS/PAGE . Integration of an insert into the yeast chromosome was confirmed by PCR amplification using AOXI primers designed to monitor the intactness of the insertion into the chromosome . The expression cassettes constructed were both expressed in E . coli strain (XL1-blue) and P . pastoris (SMD1168) respectively . An approximately 70 kDa recombinant toxin was obtained both in P . pastoris and E . coli in different quantities . Expression was confirmed by Northern-blot analysis of 2.0 kb transcripts, obtained from clones induced for RNA transcripts, which hybridized with a {(32)P}dCTP-labelled probe prepared from a 641 bp fragment of restriction-endonuclease- Hae II-digested PCR product of the cry2 gene. Int Arch Allergy Immunol, 2002 Nov, 129(3), 212 - 8 Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis; Labrada M et al.; BACKGROUND: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients . This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated . Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins . METHODS: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris . Spleen cells were fused with myeloma cells using polyethylene glycol . Hybridoma screening was performed using a direct ELISA with recombinant allergen . MAb specificity to rBlo t 13 was tested by immunoblotting . Topography of binding sites and binding of MAb to native allergen was studied by ELISA . Reactivity of MAb against allergenic extract of B . tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition . In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P . pastoris expression was compared . RESULTS: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated . These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE . In ELISA, the binding of 5G3 MAb to B . tropicalis and D . siboney extracts was inhibited by rBlo t 13 . Both MAbs showed the highest reactivity when the allergen was expressed in P . pastoris . CONCLUSION: Two MAbs specific for Blo t 13 were obtained . These MAbs recognized the same or close epitopes on the rBlo t 13 molecule . The occurrence of homologous allergens to Blo t 13 in D . siboney is suggested by the ELISA inhibition assay . Infect Immun, 2002 Dec, 70(12), 6961 - 7 Vaccination of monkeys with recombinant Plasmodium falciparum apical membrane antigen 1 confers protection against blood-stage malaria; Stowers AW et al.; A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens . To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate . Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy . We evaluated the efficacy of a form of P . falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P . falciparum parasites . Significant protection from this otherwise lethal challenge with P . falciparum was observed . Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized . The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial . The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components. Infect Immun, 2002 Dec, 70(12), 6948 - 60 In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response; Kennedy MC et al.; Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate . While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P . falciparum have been described . The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P . falciparum strains is unknown . We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies . Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P . falciparum clones . Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity . Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections . However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure . Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage. Biol Chem, 2002 Jul-Aug, 383(7-8), 1125 - 31 Cold-adapted and mesophilic brachyurins; Gudmundsdottir A; Two different types of brachyurins, termed I and II, have been described in the literature . Within type I there are two subtypes, Ia and Ib . The prototype for the type I brachyurins is Fiddler crab collagenase I . Its cold-adapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics . Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen . The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin . A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical . As a cold-adapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I . It is also more sensitive to thermal inactivation and autolysis . Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I . Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes . Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin . Substitution of a highly exposed residue in the 'autolysis loop' of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency. Biochem J, 2003 Mar 1, 370(Pt 2), 417 - 27 A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition; Crepin VF et al.; Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall . The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties . We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates . A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris . The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no . AJ293029) . The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate) . The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action. Biotechnol Bioeng, 2003 Jan 5, 81(1), 27 - 32 Biooxidation of n-hexanol by alcohol oxidase and catalase in biphasic and micellar systems without solvent; Karra-Chaabouni M et al.; Alcohol oxidase from Pichia pastoris together with catalase from bovine liver was used to oxidize n-hexanol to hexanal . For this purpose, an aqueous buffer solution was mixed with large amounts of hexanol by simple agitation, yielding a biphasic system, or by adding the nonionic surfactant Brij 35 . Initial velocities and reaction yields after 24 h were measured as a function of various parameters such as the amounts of enzymes, hexanol, or surfactant . High enzymatic activity was determined for hexanol concentrations of between 20 mass% and 80 mass% without using any additional organic solvent . The homogenization of the biphasic systems with the help of Brij 35 did not yield a significant improvement of the bioconversion, which would justify the use of surfactants . J Mol Biol, 2002 Nov 15, 324(1), 165 - 75 The disulphide mapping, folding and characterisation of recombinant Ber e 1, an allergenic protein, and SFA8, two sulphur-rich 2S plant albumins; Alcocer MJ et al.; We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris . We show that both proteins were secreted at high levels and that the purified proteins were properly folded . We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity . The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family . A model three-dimensional structure of the allergen was generated . During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation . The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 725 - 30 {High expression of a heat-stable phytase in Pichia pastoris}; Peng RH et al.; It is difficult to obtain naturally occurring phytase having the required thermostability for application in animal feeding . The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris . Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical . After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted from the cell . The strains for phytase over-expression were selected out by SDS-PAGE and enzyme analysis . After fermentation in 5 L fermention tank and induced by 0.5% methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid . The activity of phytase was 130 000 u per microlitre fluid . The thermostable phytase remained 40% active after exposure at 90 degrees for 80 min. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Nov, 34(6), 697 - 702 Increasing bioactivity of Flt3 ligand by fusing two identical soluble domains; Lu CM et al.; Flt3 ligand (FL) is a hematopoietic growth factor, initiating its in tracellular signaling cascade by binding to counterpart receptor and driving receptor dimerization . The native form of soluble FL in vivo is mainly monomeric . In this study, we constructed a rFL-FL fusion protein cDNA by linking two copies of cDNA encoding the soluble domain of FL in tandem and expressed it in Pichia pastoris . On SDS-polyacrylamide gel electrophoresis, the rFL-FL fusion protein showed a molecular weight of 43 kD, agreeing well with the predicted value . The 43 kD protein was further confirmed by Western blot using polyclonal rabbit anti-human FL antibody . The rFL-FL fusion protein exhibited about 10-fold increment in its activity on colony formation of bone marrow progenitor cells . RFL-FL fusion protein also exerted more potent effect than monomeric FL on extending the survival of starving Raji cells. FEBS Lett, 2002 Nov 6, 531(2), 265 - 72 Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity; Takai T et al.; Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1 . The active recombinant variants will be the basis for various future studies . The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity . Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested. Chin Med J (Engl), 2002 Sep, 115(9), 1352 - 7 Recombinant human heparin-binding neurite-promoting factor expressed with yeast stimulates neurites outgrowth; Wang Y et al.; OBJECTIVES: Heparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro . We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro . METHODS: cDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method . After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system . The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening . Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation . His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free . His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418 . PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome . Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol . The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth . RESULTS: In the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data . The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K . SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature . The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells . CONCLUSION: Recombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth. Protein Expr Purif, 2002 Nov, 26(2), 290 - 300 High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris; Reddy ST et al.; Mannose 6-phosphate receptors (MPRs) form essential components of the lysosomal enzyme targeting system by binding newly synthesized acid hydrolases with high (nM) affinity . We report the use of Pichia pastoris as a host to efficiently express the extracytoplasmic ligand-binding domain of the cation-dependent mannose 6-phosphate receptor . A truncated and glycosylation-deficient form of the receptor AF-Asn(81)/Stop(155) was secreted into the culture medium, yielding approximately 28mg/L after purification, which is an improvement of 10-100-fold compared to expression in baculovirus-infected insect cells and mammalian cells, respectively . Enzymatic deglycosylation indicated high-mannose sugars at the single potential glycosylation site of Asn 81 . The extent and heterogeneity of N-glycans were revealed by applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . In the case of AF-Asn(81)/Stop(155), the majority (75%) of the oligosaccharides contained chain lengths of Man(8-10)GlcNAc(2) while Man(11-12)GlcNAc(2) comprised the remaining (25%) N-linked sugars . A comparative MALDI-TOF spectra of Asn(81)/Stop(155) purified from insect cells indicated that Man(2-3)GlcNAc(2) and GlcNAcMan(2-3)GlcNAc(2) share the oligosaccharide pool . The receptor isolated from yeast was functional with respect to ligand binding and acid-dependent dissociation properties, as determined by pentamannosyl phosphate-agarose affinity chromatography . In addition, the protein was biochemically and functionally similar to Asn(81)/Stop(155) expressed in insect cells concerning its oligomeric state and binding affinity to the lysosomal enzyme, beta-glucuronidase (K(d)=1.4nM) . These results demonstrate that P . pastoris is a convenient system for the production of large quantities of functional recombinant MPRs suitable for structure-function studies. Protein Expr Purif, 2002 Nov, 26(2), 202 - 10 Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification; Paus EJ et al.; Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies . The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter . Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp . These were then integrated at the HIS locus of P . pastoris GS115 (his4) . Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s) . Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP . Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW . Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86% . Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized . The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene. Photochem Photobiol, 2002 Oct, 76(4), 457 - 61 Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris; Jorissen HJ et al.; The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris . Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively) . Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore . The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min . Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved . The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms. Yeast, 2002 Nov, 19(15), 1365 - 72 Cation/H+ antiporters mediate potassium and sodium fluxes in Pichia sorbitophila . Cloning of the PsNHA1 and PsNHA2 genes and expression in Saccharomyces cerevisiae; Banuelos MA et al.; Pichia sorbitophila grows rapidly in the presence of very high NaCl concentrations . Under these conditions, even when the K(+) concentration is low, P . sorbitophila cells can maintain low Na(+) and high K(+) contents . This remarkable capacity of P . sorbitophila fails when the external pH is not acidic . This indicates that Na(+) efflux is mediated by an electroneutral Na(+)/H(+) antiporter . We have cloned and sequenced two genes designated as PsNHA1 and PsNHA2, which probably encode two antiporters of this type . The genes present high similarity with the corresponding genes from other yeasts . The heterologous expression of PsNHA1 or PsNHA2 in a Saccharomyces cerevisiae mutant lacking the Na(+) efflux systems and sensitive to high concentrations of Na(+) and K(+) rescued the tolerance and the ability to extrude both cations . The Accession Nos of the sequenced DNA fragments are: PsNHA1, AJ496431; PsNHA2, AJ496432 . (TC 2.A.36) Appl Biochem Biotechnol, 2002 Jul-Dec, 102-103(1-6), 367 - 79 Biosynthetic activity of recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II; Bhatia Y et al.; The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii beta-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described . With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively . In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively . The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides . In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained . The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10% . Detailed kinetic analysis indicated a significant (about twofold) increase in Vmax/KM of synthetic reactions in the presence of DMSO . A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose. Biosens Bioelectron, 2002 Dec, 17(11-12), 983 - 91 Characterization of the vascular endothelial growth factor-receptor interaction and determination of the recombinant protein by an optical receptor sensor; von Tiedemann B et al.; Vascular endothelial growth factor (VEGF) is one of the most important factors controlling angiogenesis . It is a homodimeric glycoprotein belonging to the family of cysteine-knot proteins . The biological activity is transduced via membrane-spanning receptors of the tyrosine kinase receptor family . Each biologically active VEGF has two receptor binding sites leading to receptor dimerization as first step following ligand binding . The ligand-binding site of the receptor is localized on extracellular Ig-like domains . The extracellular part of the receptor Flt-1 (VEGFR-1) was expressed as soluble protein and was used as receptor in an optical affinity sensor system (BIAcore) . Suitable conditions allowed the determination of the association and dissociation rate constants as k(a)=4+/-1.2 x 10(6) M(-1) s(-1) and k(d)=3+/-0.8 x 10(-5) s(-1), respectively, leading to an affinity constant of K(D)=7.5+/-3 pM, which is within the range published already from other investigations and methods . Increasing receptor loadings of the sensor surface decreased the binding efficiency, as the ratio of bound VEGF-molecules to theoretically available binding sites increased from 1:1.5 to 1:2.6 . Increasing the surface loading further, allowed the establishment of a quantitative assay with the analytical performance being influenced by the receptor loading and the contact time between sample and immobilized receptor, i.e . sample volume . This assay was used for VEGF determination during the cultivation of a recombinant Pichia pastoris strain. Eur J Biochem, 2002 Nov, 269(21), 5295 - 302 Brassica napus soluble epoxide hydrolase (BNSEH1); Bellevik S et al.; Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses . We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique . The cDNA encodes a soluble protein containing 318 amino acid residues . The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants . A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris . The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana . BNSEH1 was shown to be a monomer by gel filtration analysis . The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1 . The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis . The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop. Biochem J, 2003 Feb 1, 369(Pt 3), 593 - 601 The C-terminal segment of the 1,3-beta-glucanase Ole e 9 from olive (Olea europaea) pollen is an independent domain with allergenic activity: expression in Pichia pastoris and characterization; Palomares O et al.; Several allergenic proteins, such as the 1,3-beta-glucanases, have been associated with plant defence responses . Ole e 9 (46 kDa) is a 1,3-beta-glucanase and major allergen from olive pollen, which is a principal cause of allergy in Mediterranean countries . Its C-terminal segment (101 amino acid residues) has been produced as a recombinant polypeptide in the yeast Pichia pastoris . The cDNA encoding the polypeptide was inserted into the plasmid vector pPICZalpha-A and overexpressed in KM71 yeast cells . The recombinant product was purified by size-exclusion chromatography followed by reversed-phase HPLC . Edman degradation, MS and CD were used to determine molecular properties of the recombinant polypeptide, which exhibited 16% alpha-helix and 30% beta-sheet as regular elements of secondary structure . Disulphide bridges of the molecule were determined at positions Cys-14-Cys-76, Cys-33-Cys-94 and Cys-39-Cys-48 . The high IgE-binding capability of the recombinant C-terminal segment of Ole e 9 against sera from Ole e 9-sensitive individuals, which was determined by immunoblotting and ELISA inhibition, supported the proper folding of the polypeptide and the maintenance of antigenic properties that it exhibits as a part of the whole allergen . These data indicated that this portion of Ole e 9 constitutes an independent domain, which could be used to study its three-dimensional structure and function, as well as for clinical purposes such as diagnosis and specific immunotherapy . Since it shows sequence similarity with portions of 1,3-beta-glucanases from plant tissues and the Gas/Phr/Epd protein families involved in yeast morphogenesis, we suggest that this domain could play an equivalent functional role within these enzymes. Di Yi Jun Yi Da Xue Xue Bao, 2002 May, 22(5), 393 - 6 Expression and purification of human endostatin in Pichia pastoris and its inhibition on the growth of mouse pulmonary adenocarcinoma cell line LA795; Xia H et al.; OBJECTIVE: To obtain yeast strain Pichia pastoris that highly expresses human endostatin by gene recombination technology, and to evaluate the inhibitory effect of recombinant human endostatin (rhES) on the growth of mouse pulmonary adenocarcinoma cell line LA795 . METHODS: The gene coding for human endostatin was cloned into the genome of Pichia pastoris through LiCl transformation method, and the clones with high soluble rhES expression were selected . Purification of rhES was performed with heparin affinity chromatography . Effects of rhES on bFGF-induced proliferation of human endothelial cell line ECV-304 cells were observed . T739 mice with subcutaneous inoculation of LA795 cells were treated with injections of either rhES or PBS for 14 consecutive days, and the volume of the tumors were measured . RESULTS: Clones with high rhES expression were obtained and purified rhES potently inhibited the proliferation of ECV-304 cells and the growth of LA795 cells in T739 mice . CONCLUSION: rhES produced by Pichia pastoris possesses good biological activities and conspicuously inhibits the growth of LA795 cells. Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 60 - 6 Epub 2002 Aug 29. Study of two-stage processes for the microbial production of 1,3-propanediol from glucose; Hartlep M et al.; The microbial production of 1,3-propanediol (1,3-PD) from glucose was studied in a two-stage fermentation process on a laboratory scale . In the first stage, glucose was converted to glycerol either by the osmotolerant yeast Pichia farinosa or by a recombinant Escherichia coli strain . In the second stage, glycerol in the broth from the first stage was converted to 1,3-PD by Klebsiella pneumoniae . The culture broth from P . farinosa was shown to contain toxic metabolites that strongly impair the growth of K . pneumoniae and the formation of 1,3-PD . Recombinant E . coli is more suitable than P . farinosa for producing glycerol in the first stage . The fermentation pattern from glycerol can be significantly altered by the presence of acetate, leading to a significant reduction of PD yield in the second stage . However, in the recombinant E . coli culture acetate formation can be prevented by fed-batch cultivation under limiting glucose supply, resulting in an effective production of 1,3-PD in the second stage with a productivity of 2.0 g l(-1) h(-1) and a high yield (0.53 g/g) close to that of glycerol fermentation in a synthetic medium . The overall 1,3-PD yield from glucose in the two stage-process with E . coli and K . pneumoniae reached 0.17 g/g. Heart Vessels, 2002 Sep, 16(6), 260 - 3 Native valve endocarditis due to Pichia ohmeri; Joao I et al.; Candida species can cause clinical manifestations in various organs of the cardiovascular system, i.e., the pericardium, myocardium, and endocardium, with endocarditis being the best-known clinical entity . Endocarditis is seen primarily in intravenous drug users and in individuals with damaged native valves, especially in congenital heart disease or rheumatic valvular diseases, and in prosthetic heart valves . The authors present a case of Pichia ohmeri endocarditis in an intravenous drug user, with an unusual presentation form . This is a case of a 42-year-old man, an intravenous heroin user, who was admitted to our Vascular Surgery Department because of fever and acute serious ischemia of the left inferior limb . He presented with fever (39 degrees C), a pale and cold left limb, absence of the left popliteal pulse, and a pansystolic murmur at the cardiac apex . The transthoracic echocardiogram showed a large vegetation on the anterior leaflet of the mitral valve and severe mitral regurgitation with good left ventricular systolic function . Empirical antibiotic therapy was started . Six days after admission, embolectomy was performed with partial clinical recovery . Three blood cultures and the embolus showed a teleomorphic form of Candida guilliermondii - Pichia ohmeri . Therapy with intravenous liposomal amphotericin B, fluocitosin, imipenem, and aztreonam was started . Two weeks later, his clinical condition deteriorated with acute heart failure refractory to medical therapy, mandating mechanical ventilation and high-dose vasopressor and inotropic amine support . He underwent urgent mitral valve replacement with a biologic prosthetic valve . Rapid stabilization of the cardiac status occurred, but ischemic limb lesions required further vascular interventions. J Biol Chem, 2002 Dec 6, 277(49), 47205 - 12 Epub 2002 Oct 08. Localization of the carbohydrate recognition sites of the insulin-like growth factor II/mannose 6-phosphate receptor to domains 3 and 9 of the extracytoplasmic region; Hancock MK et al.; The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands . Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region . Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes . Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K(d) = 0.3 +/- 0.1 nm) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d) approximately 500 nm) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3. Nat Cell Biol, 2002 Oct, 4(10), 750 - 6 De novo formation of transitional ER sites and Golgi structures in Pichia pastoris; Bevis BJ et al.; Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER . Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris . The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo . tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation . Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth . Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature. J Biochem Mol Biol, 2002 Sep 30, 35(5), 476 - 81 Secretion of Pem-CMG, a peptide in the CHH/MIH/GIH family of Penaeus monodon, in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae; Treerattrakool S et al.; The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement . In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P . monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter . In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed . The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted . The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively . The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed . This produced the mature Pem-CMG peptide. Protein Expr Purif, 2002 Oct, 26(1), 96 - 105 Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris; Sinclair G et al.; The lysosomal hydrolase glucocerebrosidase catalyzes the penultimate step in the breakdown of membrane glycosphingolipids . An inherited deficiency in this enzyme leads to the onset of Gaucher disease, the most common lysosomal storage disorder . Exogenous sources of this protein are required for biochemical and biophysical investigations and enzyme replacement therapy of Gaucher disease . Heterologous expression of glucocerebrosidase has been successful in mammalian and insect cell lines and although its use in enzyme replacement therapy of Gaucher disease has proven efficacious, current production levels limit the availability of the enzyme . Initial attempts to express human glucocerebrosidase using the methylotrophic yeast Pichia pastoris had limited success, despite significant levels of transcription . Using fragments of the glucocerebrosidase cDNA fused to the luciferase cDNA as a translational read-through reporter, the impact of synonymous codon usage bias on protein expression in P . pastoris was examined . A table of preferred codons was determined for P . pastoris and the codon usage of a 186-bp fragment of the glucocerebrosidase gene was optimized to that of the P . pastoris preferred set . A second construct with altered G+C content but no codon optimization was created for comparison . While the native glucocerebrosidase coding region limited luciferase activity to baseline levels, the codon optimized and G+C altered constructs increased luciferase activity 10.6- and 7.5-fold, respectively . Optimized G+C content, regardless of corresponding codon optimization, appears to be the major contributor to increased translational efficiency in this heterologous expression host. Protein Expr Purif, 2002 Oct, 26(1), 65 - 70 Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme; Bellevik S et al.; Epoxide hydrolases are enzymes involved in metabolism and defense of plants . Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana . To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed . We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis . We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A . thaliana . A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose . The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis . The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively . The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C . Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively . The activity was 50-fold lower towards cis-stilbene oxide . The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C. Protein Expr Purif, 2002 Oct, 26(1), 28 - 34 Expression, purification, and characterization of equine lactoferrin in Pichia pastoris; Paramasivam M et al.; Lactoferrin is an 80kDa iron-binding glycoprotein . It is secreted by exocrine glands . Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it . In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector . The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column . The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin . Its N-terminal sequence was identical to that of the native ELF . The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly . The recombinant protein appears to be hyperglycosylated by the host strain, GS115 . This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P . pastoris system . An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems. Biotechnol Bioeng, 2002 Dec 5, 80(5), 490 - 7 Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChE; Weill CO et al.; In a previous report, Morel and Massoulie showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulie, 2000) . They reported that a 5' Bungarus fragment could partially transfer this property to a CAT expression vector . This appeared to offer the possibility of increasing the production of recombinant proteins . In the present paper, we show that insertion of this fragment in the transcribed region, before the polyadenylation site, may have either stimulatory or inhibitory effects, depending on the vector and on the reporter gene . Since the stimulatory effect of Bungarus coding region could not be attached to a small number of discrete motifs, we reasoned that it might result from a general feature of the sequence . Therefore it might be possible to partially transfer this property to the very homologous human AChE (hHAChE) coding sequence by modifications based on synonymous codons, which increased nucleotide identity between the 5' fragment (721 nucleotides) of bBAChE and hHAChE from 71% to 85% . The production of human AChE in transfected COS cells was increased nearly 2-fold with this modified construct, but still remained about 4-fold smaller than that of Bungarus AChE . There was no change in expression level in transformed Pichia pastoris . We thus confirm that coding sequences can strongly influence gene expression, but in a manner that depends on the context and cannot yet be predicted . J Biol Chem, 2002 Dec 20, 277(51), 49767 - 75 Epub 2002 Sep 25. Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast; Zhang H et al.; Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D . A., Nomura, T., Talley, A . K., Cooper, R . A., Dzekunov, S . M., Ferdig, M . T., Ursos, L . M., Sidhu, A . B., Naude, B., Deitsch, K . W., Su, X . Z., Wootton, J . C., Roepe, P . D., and Wellems, T . E . (2000) Mol . Cell 6, 861-871) . Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter . Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles . Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression . Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure . We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast . To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast . Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild type versus CQR-conferring mutant Pfcrt function, which may include effects on H(+) transport (Dzekunov, S., Ursos, L . M . B., and Roepe, P . D . (2000) Mol . Biochem . Parasitol . 110, 107-124), and to perfect a rapid purification of biotinylated Pfcrt . These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of important P . falciparum transport proteins and membrane associated vaccine candidates. Curr Opin Biotechnol, 2002 Aug, 13(4), 329 - 32 Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris; Cereghino GP et al.; The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels . Using this system, recent advances have been made in the quality of recombinant proteins in fermenter culture and in the quality of the protein product, namely improved secretion signals and glycosylation patterns. Yeast, 2002 Oct, 19(14), 1203 - 20 SHAM-sensitive alternative respiration in the xylose-metabolizing yeast Pichia stipitis; Shi NQ et al.; SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood . We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption . The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum . Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool . Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose . Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis . We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism . Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source . We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P . stipitis. Yeast, 2002 Oct, 19(14), 1191 - 202 Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris; Hirose M et al.; We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris . rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial . The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT) . In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns . The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated . The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components . The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column . Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components . The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components . Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels . Mol Biochem Parasitol, 2002 Aug 28, 123(2), 125 - 34 A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis; Hussein AS et al.; A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species . Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M) . All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs . Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans . Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine . A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine . This model is consistent with mutational analysis of the nematode enzymes . Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms . These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity . J Biotechnol, 2002 Oct 23, 99(2), 97 - 110 Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen; Bisht H et al.; A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris . Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates . Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs) . Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein . Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data . J Biol Chem, 2002 Nov 15, 277(46), 44035 - 43 Epub 2002 Aug 30. Specific characterization of substrate and inhibitor binding sites of a glycosyl hydrolase family 11 xylanase from Aspergillus niger; Tahir TA et al.; The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis . Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined . The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced . The low specific activities of Y6A and Y89A were entirely accounted for by a change in k(cat) and K(m), respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K(m) and decreased k(cat) . Tyr(6), Tyr(10), Tyr(89), Tyr(164), and Trp(172) are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A . niger xylanase in complex with the modeled xylobiose . All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity . Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism . The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase . The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration . A close inspection of the three-dimensional structure of A . niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases. Eur J Biochem, 2002 Sep, 269(18), 4586 - 96 Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier; Briand L et al.; Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication . We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein . The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide . ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing . The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs . CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs . Gel filtration analysis showed that ASP3c is monomeric at neutral pH . Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range . It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual) . This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant. Infect Immun, 2002 Oct, 70(10), 5676 - 83 Secreted metalloprotease gene family of Microsporum canis; Brouta F et al.; Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors . Microsporum canis, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported . Using Aspergillus fumigatus metalloprotease genomic sequence (MEP) as a probe, three genes (MEP1, MEP2, and MEP3) were isolated from an M . canis genomic library . They presented a quite-high percentage of identity with both A . fumigatus MEP and Aspergillus oryzae neutral protease I genes . At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these M . canis genes (MEP genes) encode a zinc-containing metalloprotease gene family . Furthermore, MEP3 was found to be the gene encoding a previously isolated M . canis 43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in Pichia pastoris . Reverse transcriptase nested PCR performed on total RNA extracted from the hair of M . canis-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process . This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes. J Clin Oncol, 2002 Sep 15, 20(18), 3792 - 803 Phase I study of recombinant human endostatin in patients with advanced solid tumors; Herbst RS et al.; PURPOSE: Endostatin, a 20-kd fragment of collagen XVIII, is a potent inhibitor of angiogenesis . We evaluated recombinant human endostatin (rh-Endo) in a phase I trial designed to assess safety, pharmacokinetics, and serum markers of angiogenesis in patients with solid tumors . PATIENTS AND METHODS: Twenty-six patients were enrolled onto a dose-finding trial of rh-Endo administered as an intravenous bolus over a 20-minute period once daily . Three patients each were treated at dose le