Mar Biotechnol (NY), 2003 Jul-Aug, 5(4), 373 - 9 Expression of biologically active crustacean hyperglycemic hormone (CHH) of Penaeus monodon in Pichia pastoris; Treerattrakool S et al.; Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans . This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction . In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter . The recombinant Pem-CMG was secreted into the culture medium using the alpha-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide . The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing . The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity . The final yield of the recombinant Pem-CMG after purification was 260 micro g/L of the culture medium . Both crude and purified recombinant Pem-CMG produced from P . pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P . monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P . monodon. J Gen Virol, 2004 Jan, 85(Pt 1), 241 - 9 Expression of functionally active helper component protein of Tobacco etch potyvirus in the yeast Pichia pastoris; Ruiz-Ferrer V et al.; Tobacco etch potyvirus (TEV) is transmitted by aphids in a non-persistent manner with the assistance of a virus-encoded protein known as helper component (HC-Pro) . To produce a biologically active form of recombinant TEV HC-Pro protein, heterologous expression in the methylotrophic yeast Pichia pastoris was used . A cDNA encoding the TEV HC-Pro region, fused to a Saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, was inserted into the P . pastoris genome using a modified version of the pPIC9 vector . The expressed TEV HC-Pro protein was obtained directly from culture medium of recombinant yeast colonies; it was able to interact with TEV particles in a protein overlay binding assay, and also to assist aphid transmission of purified TEV particles to plants using the aphid Myzus persicae as vector . Our results indicate that P . pastoris provides a rapid and low-cost heterologous expression system that can be used to obtain biologically active potyvirus HC-Pro protein for in vitro transmission assays. Protein Expr Purif, 2004 Feb, 33(2), 185 - 94 Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris; McKinney J et al.; Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria . However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme . We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols . When expressed as a fusion protein in E . coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins . The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P . pastoris . To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated . Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme . Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme . This supports previous observations that the enzyme in vivo may be stabilized by additional interactions. Protein Expr Purif, 2004 Feb, 33(2), 176 - 84 Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris; de Jong LA et al.; The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties . The dopamine antagonist {3H}spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor . The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin . The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction . A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively. J Biotechnol, 2004 Jan 22, 107(2), 115 - 24 Synthesis of the measles virus nucleoprotein in yeast Pichia pastoris and Saccharomyces cerevisiae; Slibinskas R et al.; The development of a simple, efficient and cost-effective system for generation of measles virus nucleoprotein might help to upgrade reagents for measles serology . The gene encoding measles nucleoprotein was successfully expressed in two different yeast genera, Pichia pastoris and Saccharomyces cerevisiae, respectively . Both yeast genera synthesized a high level of nucleoprotein, up to 29 and 18% of total cell protein, in P . pastoris and S . cerevisiae, respectively . This protein is one of most abundantly expressed in yeast . After purification nucleocapsid-like particles (NLPs) derived from both yeast genera appeared to be similar to those detected in mammalian cells infected with measles virus . A spontaneous assembly of nucleoprotein into nucleocapsid-like particles in the absence of the viral leader RNA or viral proteins has been shown . Compartmentalisation of recombinant protein into large compact inclusions in the cytoplasm of yeast S . cerevisiae by green fluorescence protein (GFP) fusion has been demonstrated . Sera from measles patients reacted with the recombinant protein expressed in both yeast genera and a simple diagnostic assay to detect measles IgM could be designed on this basis. Eur J Pharm Sci, 2004 Jan, 21(1), 61 - 7 Analysis of the transport properties of side chain modified dipeptides at the mammalian peptide transporter PEPT1; Knutter I et al.; This study was initiated to examine systematically the effect of side chain modifications at dipeptides on their transport via PEPT1 . We synthesized a series of Xaa(R)-Ala and Ala-Xaa(R) dipeptides with the functional groups of the side chains modified by structurally different blocking groups R . Recognition and transport of these derivatives by PEPT1 was measured in Caco-2 cells, in transgenic Pichia pastoris cells and in Xenopus laevis oocytes expressing PEPT1 . The dipeptide derivatives displayed K(i) values between 0.002 and 4 mM . Electrophysiological analyses showed that the Ala-Xaa(R) derivatives were transported by PEPT1 . In contrast, most Xaa(R)-Ala derivatives--although recognized--did not show significant transport rates . Substitution of a terminal phenyl residue in the side chain blocking group by a p-nitrophenyl residue enhanced the affinity of several dipeptide derivatives for interaction with PEPT1 . However, none of these compounds showed electrogenic transport in oocytes . With a K(i) value of 0.002 mM, Lys{Z(NO(2))}-Val displayed the highest affinity to PEPT1 ever reported . We conclude that the transport of side chain modified dipeptides into enterocytes depends (a) on the position of the modified trifunctional amino acid in the dipeptide, (b) the distance between its alpha-carbon and the side chain blocking group and (c) the hydrophobic character of the side chain modification. J Protein Chem, 2003 Aug, 22(6), 509 - 14 Kinetic analysis of the binding of hemopexin-like domain of gelatinase B cloned and expressed in Pichia pastoris to tissue inhibitor of metalloproteinases-1; Stute J et al.; The gelatinases are a subgroup of the matrix metalloproteinase family . The interaction of their C-terminal hemopexin-like domain with a tissue inhibitor of metalloproteinases (TIMP) is a major part of the regulatory mechanisms of gelatinases . To investigate the interaction of the hemopexin-like domain of gelatinase B (92-Pex) and TIMP-1, we expressed the individual domain in Pichia pastoris . The active refolded domain was purified by ion exchange chromatography and gel filtration . We investigated the formation of the 92-Pex/TIMP-1 complex by surface plasmon resonance (SPR) . The dissociation constant Kd was calculated to be 0.86 nM . Analogous to the complex of the hemopexin-like domain of gelatinase A and TIMP-2 (Olson, M . W . et al., 1997), the binding curves of the 92-Pex/TIMP-1 complex were best fitted with a monophasic model. Biochem Biophys Res Commun, 2004 Jan 16, 313(3), 534 - 40 Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases; Kim JS et al.; Apolipoprotein(a) {apo(a)} contains the largest numbers of kringle domains identified to date . Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor . To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined . rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner . This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions . rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay . These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo. Zhonghua Gan Zang Bing Za Zhi, 2003 Dec, 11(12), 728 - 30 {The primary study on the anti-HBV effect of whole recombinant yeast}; Zeng Y et al.; OBJECTIVES: Based on the immunologic character of Pichia pastoris yeast, a new therapeutic vaccine, whole recombinant yeast, was used to explore a new way to activate cell-mediated anti-viral immunity . METHODS: The recombinant plasmids, pPIC9K/S and PIC9K/hsp(1-370)-S, were constructed by inserting the gene encoding HBsAg, HSP70 (1-370) -HBsAg into vector pPIC9K and then the recombinants were transfected into Pichia pastoris yeast,GS115, respectively . Then that recombinant yeast immunized BALB/C mice were detected for humoral and cellular immunity to HBsAg . RESULTS: Recombinant yeast successfully activated the humoral immunity to HBsAg in mice, but failed to activate the cellular immunity . CONCLUSION: The whole recombinant yeast can be used as vaccine, but need further study for optimal way of immunization. Cancer Res, 2003 Dec 15, 63(24), 9032 - 41 Human kallikrein 14: a new potential biomarker for ovarian and breast cancer; Borgono CA et al.; Human kallikrein gene 14 (KLK14) is a recently discovered member of the tissue kallikrein family of secreted serine proteases, which includes hK3/prostate-specific antigen, the best cancer biomarker to date . Given that KLK14 is hormonally regulated, differentially expressed in endocrine-related cancers, and a prognostic marker for breast and ovarian cancer at the mRNA level, we hypothesize that its encoded protein, hK14, like hK3/prostate-specific antigen, may constitute a new biomarker for endocrine-related malignancies . The objective of this study was to generate immunological reagents for hK14, to develop an ELISA and immunohistochemical techniques to study its expression in normal and cancerous tissues and biological fluids . Recombinant hK14 was produced in Pichia pastoris, purified by affinity chromatography, and injected into mice and rabbits for polyclonal antibody generation . Using the mouse and rabbit antisera, a sandwich-type immunofluorometric ELISA and immunohistochemical methodologies were developed for hK14 . The ELISA was sensitive (detection limit of 0.1 micro g/liter), specific for hK14, linear from 0 to 20 micro g/liter with between-run and within-run coefficients of variation of <10% . hK14 was quantified in human tissue extracts and biological fluids . Highest levels were observed in the breast, skin, prostate, seminal plasma, and amniotic fluid, with almost undetectable levels in normal serum . hK14 concentration was higher in 40% of ovarian cancer tissues compared with normal ovarian tissues . Serum hK14 levels were elevated in a proportion of patients with ovarian (65%) and breast (40%) cancers . Immunohistochemical analyses indicated strong cytoplasmic staining of hK14 by the epithelial cells of normal and malignant skin, ovary, breast, and testis . In conclusion, we report the first ELISA and immunohistochemical assays for hK14 and describe its distribution in tissues and biological fluids . Our preliminary data indicate that hK14 is a potential biomarker for breast and ovarian cancers. Glycobiology, 2004 Mar, 14(3), 265 - 74 Epub 2003 Dec 23. Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris; Trimble RB et al.; Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology . The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc . hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al . {1991}, J . Biol . Chem., 266, 22807-22817) . The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites . Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis . The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol . Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol . Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon . This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P . pastoris. Glycobiology, 2004 May, 14(5), 417 - 29 Epub 2003 Dec 23. Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris; Yoshimasu MA et al.; A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin . The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris . All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type . Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered . Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0 . Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile . Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability . Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type . It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein. Gene, 2004 Jan 7, 324, 129 - 37 Cloning and characterization in Pichia pastoris of PNO1 gene required for phosphomannosylation of N-linked oligosaccharides; Miura M et al.; The yeast Pichia pastoris PNO1 (Phosphomannosylation of N-linked Oligosaccharides) gene, which is involved in phosphomannosylation of N-linked oligosaccharides, was cloned using the Saccharomyces cerevisiae MNN4 gene {Glycobiology 6 (1996) 805} as a probe . The PNO1 open reading frame (ORF) encodes a type II membrane protein composed of 777 amino acid residues . Only in the short region extending from amino acid position 450 to 606 of Pno1p, sequence homology to S . cerevisiae Mnn4p was observed at a level of 45% . The tandem repeat sequence of Lys-Lys-Lys-Lys-Glu-Glu-Glu-Glu characteristic of the C-terminal region of S . cerevisiae Mnn4p is not present in Pno1p . To investigate the function of the PNO1 gene, we constructed a PNO1 gene disruptant by replacement with an expression cassette of human antithrombin (AT), a glycoprotein in plasma . The cell growth and recombinant human antithrombin (rAT) production levels of the disruptant were similar to those of recombinant human antithrombin-expressing wild-type strains . Moreover, the level of alcian blue dye cell staining, which shows the presence of acidic sugar chains on the cell surface, was also similar . However, the phosphomannosylation ratio of N-linked oligosaccharides on recombinant human antithrombin decreased dramatically from 20% in wild-type strains to less than 1% in the PNO1 disruptant . When the PNO1 gene was re-introduced into the disruptant, the phosphomannosylation ratio recovered to the original level . These results suggest that the newly cloned PNO1 gene promotes phosphomannosylation only to core-like oligosaccharides, and not to the hypermannosylated outer chain, and that it has a different function from the MNN4 gene, which promotes the phosphomannosylation of both core and outer sugar chains. Biochemistry, 2003 Dec 30, 42(51), 15148 - 57 The crystal structure of Pichia pastoris lysyl oxidase; Duff AP et al.; Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides . Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta . Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes . The structure of PPLO has been determined at 1.65 A resolution . PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue . While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible . The structural elements that are responsible for the accessibility of the active site are identified and discussed. Protein Expr Purif, 2003 Nov, 32(1), 28 - 34 High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris; Ouyang J et al.; Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition . Pichia pastoris was used as a host to efficiently express the pST gene in this study . Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures . SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST . The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography . MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size . Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2 . Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection . These results indicate that the P . pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels. J Biol Chem, 2004 Mar 5, 279(10), 8577 - 84 Epub 2003 Dec 16. Glycodelin A, not glycodelin S, is apoptotically active . Relevance of sialic acid modification; Mukhopadhyay D et al.; Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles . The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans . It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells . The precise role of GdS in seminal plasma is not known . In this study, we report that GdS is not apoptotically active . We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated . We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form . Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms. Biotechnol Lett, 2003 Nov, 25(21), 1795 - 800 On-line monitoring of the methanol concentration in Pichia pastoris cultures producing an heterologous lipase by sequential injection analysis; Surribas A et al.; An automated sequential injection analysis (SIA) system using stop-flow technique was developed to determine methanol concentration by means of the enzymatic reactions of alcohol oxidase and peroxidase . Its application as an on-line device for monitoring Pichia pastoris fermentations producing an heterologous protein was demonstrated . Linear response, observed up to 2 g l(-1), was reached by including a dilution chamber in the SIA manifold . The sampling frequency was 7 analyses per hour with a relative standard deviation lower than 4%. Yeast, 2003 Dec, 20(16), 1331 - 7 Cloning and sequence analysis of the TRP1 gene encoding the phosphoribosyl anthranilate isomerase from Pichia anomala (strain K); Friel D et al.; Pichia anomala (strain K) is an efficient biocontrol agent against post-harvest diseases affecting apples . To study the role of strain K genes in biocontrol activity, it is useful to identify selectable markers on which to base a gene disruption strategy . The Pichia anomala TRP1 gene (PaTRP1) was isolated by complementation of the multi-auxotrophic S . cerevisiae strain FY-1679-18b . DNA sequence analysis revealed the presence of a 699 bp ORF encoding a 233 amino acid protein showing the typical conserved structure of proteins of the phosphoribosyl anthranilate isomerase (PRAI) family . Codon analysis revealed a high number of unused codons . Downstream from PaTRP1 was found the 3' extremity of a gene highly similar to the IPP1 gene (coding for the inorganic pyrophosphatase) . J Plant Physiol, 2003 Nov, 160(11), 1385 - 91 Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne); Chalmers J et al.; A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated . The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns . Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids . Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains . Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths . The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity. Parasitol Res, 2004 Jan, 92(2), 173 - 6 Epub 2003 Dec 04. Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris; Fong MY et al.; A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris . Specificity of the recombinant TES-120 antigen produced by the yeast was investigated . Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays . Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only . This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis. Poult Sci, 2003 Nov, 82(11), 1726 - 32 Efficacy of a phytase derived from Escherichia coli and expressed in yeast on phosphorus utilization and bone mineralization in turkey poults; Applegate TJ et al.; A slope-response bioassay was conducted with male turkey poults to determine the sparing effect of P, based on improvements in bone mineralization in turkey poults, from 10 to 21 d of age when diets were supplemented with a novel phytase . Reference diets for calculation of the sparing effect of P contained 0.47, 0.55, 0.70, and 0.79% nonphytate phosphorus (NPP) . Diets with varying dosages of a swine, Escherichia coli-derived AppA2 phytase (ECP) expressed in Pichia pastoris yeast (0, 250, 500, 750, and 1,000 U/kg) were added to the 0.47% NPP diet and improvements in bone mineralization determined the sparing effect of P supplied from ECP . Two additional reference diets were included that contained 500 U/kg from one of two commercial phytases (PA and PB) derived from Aspergillus and Peniophora . At 500 U/kg diet the ECP spared an additional 0.22% NPP (if calculated from tibia ash %), 0.18% NPP (if calculated from toe ash %), 0.24% NPP (if calculated from mg tibia ash), or 0.21% NPP (if calculated from mg toe ash) . Phosphorus retention results validate bioassay results, in that 500 U ECP/kg resulted in 68.2% P being retained (0.49% of diet P retained) as compared with only 58.9% P being retained from the unsupplemented control diet (0.421% of diet P retained; P < 0.05). Biosci Biotechnol Biochem, 2003 Nov, 67(11), 2334 - 43 Functional properties of glycosylated lysozyme secreted in Pichia pastoris; Saito A et al.; Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system . The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter) . Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis . The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32) . The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable . The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme. J Biotechnol, 2003 Dec 5, 106(1), 53 - 68 Macrokinetic model for methylotrophic Pichia pastoris based on stoichiometric balance; Ren HT et al.; A macrokinetic model for Pichia pastoris expressing recombinant human serum albumin is proposed . The model describes the balances of some key metabolites, ATP and NADH, during glycerol and methanol metabolism . In the glycerol growth phase, the metabolic pathways mainly include phosphorylation, glycolysis, tricarboxylic acid cycle, and respiratory chain . In the methanol growth phase, methanol is oxidized to formaldehyde at first . Then, while a part of formaldehyde is oxidized to formate, the rest is condensed with xylulose-5-monophosphate to form glyceraldehyde-3-phosphate, and further assimilated to form cell constituents . The metabolic pathways following glyceraldehyde-3-phosphate were assumed to be similar to those in the glycerol growth phase . Based on the model, the macrokinetic bioreaction rates such as the specific substrate consumption rate, the specific growth rate, the specific acetyl-CoA formation rate as well as the specific oxygen uptake rate are obtained . The specific substrate consumption rate and the specific growth rate are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, i.e., the medium volume, the concentrations of the biomass, the substrate, and the product, is set up . Experimental results demonstrate that the model can describe the cell growth and the protein production with reasonable accuracy. Gen Comp Endocrinol, 2003 Dec, 134(3), 244 - 54 Expression of a biologically active recombinant follicle stimulating hormone of Japanese Eel Anguilla japonica using methylotropic yeast, Pichia pastoris; Kamei H et al.; In the Japanese eel Anguilla japonica, the administration of exogenous GTH is necessary for the artificial induction and completion of gonadal maturation due to its GTH deficiency under captive conditions . The isolation of native eel GTH has not been accomplished, which has made it difficult to fully elucidate the biological functioning of the two GTHs (FSH and LH) in eel . In this study, we attempted to produce a recombinant Japanese eel GTH (rjeFSH) having biological activity using methylotrophic yeast, Pichia pastoris in order to gain more understanding of the functioning of GTH in this species . An expression vector in which jeFSHbeta and GTHalpha subunit cDNAs were tandemly connected was constructed . P . pastoris was transformed with the vector, and rjeFSH was expressed . The rjeFSH thus expressed was detected by Western blot analysis . The glycoprotein fraction of the yeast culture supernatant was separated by native PAGE, and a band showed positive reaction with anti-GTHalpha and FSHbeta antisera similarly, suggesting that both subunits are associated . After deglycosylation, both subunits were decreased in molecular mass, indicating that rjeFSH was glycosylated . In in vitro assay, rjeFSH stimulated the release of testosterone and 11-ketotestosterone from immature eel testis, whereas release was not stimulated in maturing eel testis . This is the first report investigating the biological activity of eel GTH using the recombinant eel FSH. Mol Ecol, 2003 Nov, 12(11), 3137 - 45 Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts; Suh SO et al.; During a survey of insect gut micro-organisms, we consistently isolated Pichia stipitis-like yeasts (Fungi: Ascomycota, Saccharomycetes) from the wood-ingesting beetles, Odontotaenius disjunctus and Verres sternbergianus (Coleoptera: Passalidae) . The yeasts were isolated from passalid beetles over a wide area, including the eastern and midwestern USA and Panama . Phylogenetic analyses of the nuclear encoded small and large subunit rRNA gene (rDNA) sequences distinguished a well-supported clade consisting of the passalid yeasts and Pichia stipitis, P . segobiensis, Candida shehatae and C . ergatensis . Members of this clade have the ability to ferment and assimilate xylose or to hydrolyse xylan, major components of the polysaccharide, hemicellulose . Sexual reproduction was present in the passalid isolates but was rare among the gut yeasts of other beetles to which they were compared . Minor genetic and phenotypic variation among some of the passalid yeasts was detected using markers from the internal transcribed spacer region of the rDNA repeat unit, morphology, and in vitro metabolic tests . The consistent association of xylose-fermenting yeasts of almost identical genotypes with passalid beetles across a broad geographical distribution, suggests a significant symbiotic association. Biochem J, 2004 Mar 1, 378(Pt 2), 657 - 63 Expression and characterization of cathepsin P; Mason RW et al.; The mouse genome contains a family of clan C1A proteases that appear to be restricted to rodents within Eutherian (placental) mammals . mRNA analysis has shown that these genes are expressed exclusively in placenta . Sequence analysis predicts that the expressed proteins will be functional and consequently it was proposed that this family of proteases may have evolved to perform subspecialized functions of the closely related protease, cathepsin L, that is expressed in placental tissues of all mammalian species . In the present study, it was shown that cathepsin P can be expressed in Pichia pastoris as an inactive zymogen that can be activated with proteinase K, chymotrypsin or pancreatic elastase at neutral pH . Unlike other mammalian cathepsins, cathepsin P could also be autoactivated at neutral pH, but not at acidic pH . The activated enzyme was capable of hydrolysing peptidyl substrates and the protein substrates azocasein and transferrin, with optimal activity at pH 6.5-7.5 . Little activity could be detected at pH 5.0 and below . Salts such as Na2SO4 and hyaluronate stimulated the activity of the protease against peptidyl substrates . The properties of cathepsin P appear to be quite distinct from those of cathepsin L, indicating that the duplication that gave rise to cathepsin P has probably not yielded an enzyme that provides a subfunction of cathepsin L in rodents . It seems probable that cathepsin P has evolved to perform a function that is performed by an evolutionarily unrelated protease in other mammalian species. J Virol Methods, 2003 Dec, 114(2), 145 - 50 Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA; Ao JQ et al.; Pseudorabies virus glycoprotein E (PRV gE) has been recognized as a suitable diagnostic antigen for pseudorabies . In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system . SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins, approximately 38 and 32 kDa, in the culture supernatant of P . pastoris integrant 72 h after induction . Protein concentration assay showed the expression product amounted to 106.7 mg/l, accounting for 66.67% of total culture supernatant proteins . An indirect PRV gE-ELISA was then established by using the recombinant expression product as a coating antigen . Cross-reactivity assay showed that this antigen was PRV specific . Reproducibility experiment displayed good consistency . Comparison of detection results of 348 field serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed there was no significant difference between these two methods (P > 0.05). Adv Drug Deliv Rev, 2003 Nov 28, 55(12), 1547 - 67 Recombinant collagen and gelatin for drug delivery; Olsen D et al.; The tools of recombinant protein expression are now being used to provide recombinant sources of both collagen and gelatin . The primary focus of this review is to discuss alternatives to bovine collagen for biomedical applications . Several recombinant systems have been developed for production of human sequence collagens . Mammalian and insect cells were initially used, but were thought to be too costly for commercial production . Yeast have been engineered to express high levels of type I homotrimer and heterotrimer and type II and type III collagen . Co-expression of collagen genes and cDNAs encoding the subunits of prolyl hydroxylase has lead to the synthesis of completely hydroxylated, thermostable collagens . Human types I and III collagen homotrimers have been expressed in transgenic tobacco plants, while transgenic mice have been engineered to produce full-length type I procollagen homotrimer as well as a alpha2 (I) homotrimeric mini-collagen . Most recently, a transgenic silkworm system was used to produce a fusion protein containing a collagenous sequence . Each of these transgenic systems holds great promise for the cost-effective large-scale production of recombinant human collagens . As seen in other recombinant expression systems, transgenic silkworms, tobacco, and mice lack sufficient endogenous prolyl hydroxylase activity to produce fully hydroxylated collagen . In mice and tobacco, this was overcome by over-expression of prolyl hydroxylase, analogous to what has been done in yeast and insect cell culture . In addition to recombinant alternatives to bovine collagen, other sources such as fish and sponge collagen are discussed briefly . Recombinant gelatin has been expressed in Pichia pastoris and Hansenula polymorpha in both non-hydroxylated and hydroxylated forms . Pichia was shown to be a highly productive system for gelatin production . The recombinant gelatins produced in yeast are of defined molecular weight and physio-chemical properties and represent a new biomaterial not previously available from animal sources . Genetic engineering has made great progress in the areas of recombinant collagen and gelatin expression, and there are now several alternatives to bovine material that offer an enhanced safety profile, greater reproducibility and quality, and the ability of these materials to be tailored to enhance product performance. Yeast, 2003 Nov, 20(15), 1279 - 90 Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris; Nett JH et al.; A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P . pastoris URA5 and SEC65 genes . Using additional information from a partial genomic sequence of P . pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene . A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene . The P . pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified . To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1 . The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid . We also assembled a set of modular plasmids that can be used for the stable genetic modification of P . pastoris via a double cross-over event . The sequence presented here has been submitted to the EMBL data library under Accession No . AY303544 . Malar J . 2003 Nov 14;2(1):39. Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1; Rodrigues MH et al.; BACKGROUND: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals . Because P . vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used . Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P . vivax (MSP119) could be useful for serological detection of malaria infection . METHODS: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P . vivax infection . The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria . RESULTS: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95% . The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively . The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE) . CONCLUSIONS: Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P . vivax malaria. Plant J, 2003 Dec, 36(5), 697 - 710 Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.); Van den Ende W et al.; About 15% of flowering plant species synthesize fructans . Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated . This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans . The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described . Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots . Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning . Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded . Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases . Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely . The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris . As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious . Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria. Arterioscler Thromb Vasc Biol, 2004 Jan, 24(1), 147 - 54 Epub 2003 Nov 13. Reduction of atherosclerotic plaques by lysosomal acid lipase supplementation; Du H et al.; OBJECTIVE: Proof of principle is presented for targeted enzyme supplementation by using lysosomal acid lipase to decrease aortic and coronary wall lipid accumulation in a mouse model of atherosclerosis . METHODS AND RESULTS: Mice with LDL receptor deficiency were placed on an atherogenic diet and developed predictable aortic and coronary atheroma . alpha-Mannosyl-terminated human lysosomal acid lipase (phLAL) was produced in Pichia pastoris, purified, and administered intravenously to such mice with either early or late lesions . phLAL injections reduced plasma, hepatic, and splenic cholesteryl esters and triglycerides in affected mice . phLAL was detected in hepatic Kupffer cells and in atheromatous foam cells . Repeated enzyme injections were well tolerated, with no obvious adverse effects . In addition, the coronary and aortic atheromatous lesions were (1) eliminated in their early stages and (2) quantitatively and qualitatively reduced in their advanced stages . CONCLUSIONS: These results support the potential utility of lysosomal acid lipase supplementation for the treatment of atherosclerosis, a leading cause of mortality and morbidity in Westernized nations. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 1035 - 9 {Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris}; Yu QW et al.; To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report . rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above . The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA . The molecular weight and specificity were demonstrated by PAGE and Western blot . The density of the specific precipitation bands was determined by gel scanning . The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI) . The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant . The molecular weight was 22 kD . The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively . The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively . The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L . The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC . The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity . The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale. FEMS Yeast Res, 2003 Nov, 4(2), 185 - 93 Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element; Klabunde J et al.; A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H . polymorpha-derived integration sequence . The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study . As in H . polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene . Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae . Based on these results the design of a 'universal' fungal vector appears to be feasible. FEMS Yeast Res, 2003 Nov, 4(2), 157 - 64 Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha; Ryabova OB et al.; Wild-type strains of the thermotolerant methylotrophic yeast Hansenula polymorpha are able to ferment glucose, cellobiose and xylose to ethanol . H . polymorpha most actively fermented sugars to ethanol at 37 degrees C, whereas the well-known xylose-fermenting yeast Pichia stipitis could not effectively ferment carbon substrates at this temperature . H . polymorpha even could ferment both glucose and xylose up to 45 degrees C . This species appeared to be more ethanol tolerant than P . stipitis but more susceptible than Saccharomyces cerevisiae . A riboflavin-deficient mutant of H . polymorpha increased its ethanol productivity from glucose and xylose under suboptimal supply with riboflavin . Mutants of H . polymorpha defective in alcohol dehydrogenase activity produced lower amounts of ethanol from glucose, whereas levels of ethanol production from xylose were identical for the wild-type strain and the alcohol dehydrogenase-defective mutant. Nahrung, 2003 Oct, 47(5), 359 - 63 Expression and characterization of chicken ovoinhibitor in Pichia pastoris; Begum S et al.; Chicken ovoinhibitor cDNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using chicken oviduct mRNA . The ovoinhibitor cDNA was successfully cloned downstream from the AOXI promoter of pPICZalphaA plasmid vector to facilitate its expression in the methylotrophic yeast Pichia pastoris . The pPICZalphaA carrying the ovoinhibitor cDNA was integrated into the Pichia genome . The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAE sepharose column . The recombinant ovoinhibitor had a molecular mass of 49 kDa, as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and time of flight-mass spectrometry (TOF-MS) analyses . The recombinant ovoinhibitor, just as the native ovoinhibitor, showed inhibitory activity against trypsin, chymotrypsin and elastase. Prep Biochem Biotechnol, 2003 Nov, 33(4), 269 - 81 Expression of kringle 5 domain of human plasminogen in Pichia pastoris; Zhu M et al.; The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation . It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy . It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies . When expressed in E . coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low . In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression . On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture . After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture. J Ind Microbiol Biotechnol, 2003 Nov, 30(11), 643 - 50 Epub 2003 Nov 06. Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths; Plantz BA et al.; The ability to detect viable contaminants in cultures propagated from the original host-expression system ensures that the integrity and purity of seed banks, fermentation broths, and ultimately the final product are continually controlled and maintained . The method developed to detect such agents must be selective for a broad spectrum of microbes, which may be present at very low levels, while discriminating from the host organisms . Although Pichia pastoris strains are frequently used as cell lines for the expression of heterologous proteins, a method that is specific for monitoring culture purity has yet to be reported for this type of organism . An assay that is capable of recovering contaminating bacteria, fungi, and closely related yeast from cultures of P . pastoris at parts per million detection limits is described here. J Biol Chem, 2004 Feb 6, 279(6), 3900 - 5 Epub 2003 Nov 06. Defensins from insects and plants interact with fungal glucosylceramides; Thevissen K et al.; Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations . In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2 . GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide . In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P . pastoris but not with soybean nor human glucosylceramides . Furthermore, the P . pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization . A model for the mode of action of RsAFP2 is presented in which all of these findings are linked . Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C . albicans and P . pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species . Furthermore, heliomicin interacts with glucosylceramides isolated from P . pastoris and soybean but not with human glucosylceramides . These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor. Appl Microbiol Biotechnol, 2004 Apr, 64(3), 403 - 9 Epub 2003 Nov 05. Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation; Fredlund E et al.; The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions . Growth and metabolite formation of P . anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis . Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively . HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step . Biomass production was higher in treatment (A), whereas the specific ethanol production rate during growth on glucose was similar in both treatments . This implies that oxygen availability affected the respiration and not the fermentation of the yeast . Following glucose depletion, ethanol was oxidized to acetate in treatment (A), but continued to be produced in (B) . Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (B) . Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments . The levels of these metabolites were generally significantly higher in treatment (B) than in (A), indicating their importance for P . anomala during severe oxygen limitation/anaerobic conditions. Chem Pharm Bull (Tokyo), 2003 Nov, 51(11), 1237 - 40 Identification and biological activity of microbial metabolites of xanthohumol; Herath W et al.; Microbial transformation of xanthohumol using the culture broth of Cunninghamella echinulata NRRL 3655 afforded (2S)-8-{4"-hydroxy-3"-methyl-(2"-Z)-butenyl}-4',7-dihydroxy-5-methoxyflavanone (5) and (2S)-8-{5"-hydroxy-3"-methyl-(2"-E)-butenyl}-4',7-dihydroxy-5-methoxyflavanone (6) . Xanthohumol (1) and flavanone 6 as well as (E)-2"-(2"'-hydroxyisopropyl)-dihydrofurano{2",3":4',3'}-2',4-dihydroxy-6'-methoxychalcone (2), (2S)-2"-(2"'-hydroxyisopropyl)-dihydrofurano{2",3":7,8}-4'-hydroxy-5-methoxyflavanone (3) obtained with Pichia membranifaciens showed antimalarial activity against Plasmodium falciparum. Protein Eng, 2003 Oct, 16(10), 761 - 70 Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation; Yang K et al.; The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable . We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies . Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L) . High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification . Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed . Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer . Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs . Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies. Mol Biochem Parasitol, 2003 Dec, 132(2), 83 - 92 Cloning and expression of two secretory acetylcholinesterases from the bovine lungworm, Dictyocaulus viviparus; Lazari O et al.; We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus . The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another . The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans . Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C . elegans ACE-2 over 577 amino acids, respectively . The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site . Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct . When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics . Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity . The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases. Biochemistry, 2003 Nov 11, 42(44), 13049 - 57 All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands; Croy JE et al.; The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP) . Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography . Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter . Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands . Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments . As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451) . All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor . All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes . Only sLRP4 bound thrombin-antithrombin III complexes . The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P . pastoris and readily purified . Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP. Appl Microbiol Biotechnol, 2004 Feb, 63(5), 567 - 70 Epub 2003 Nov 01. Identification of a type-D feruloyl esterase from Neurospora crassa; Crepin VF et al.; Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries . In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa . We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases . This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity . Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials . The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase. Appl Microbiol Biotechnol, 2004 Feb, 63(5), 495 - 509 Epub 2003 Nov 01. Metabolic engineering for improved fermentation of pentoses by yeasts; Jeffries TW et al.; The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast . Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified . Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production . Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation . Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae . Anaerobic growth by recombinant mutants has been reported . Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production . Recently, two routes for arabinose metabolism have been engineered in S . cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications. J Biol Chem, 2004 Feb 6, 279(6), 4459 - 64 Epub 2003 Oct 31. Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L; Lartigue A et al.; Pheromone binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in several sensory organs from moth and other insect species . They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors . We report here the crystal structure of a PBP (Amel-ASP1) originating from the honey-bee (Apis mellifera) antennae and expressed as recombinant protein in the yeast Pichia pastoris . Crystals of Amel-ASP1 were obtained at pH 5.5 using the nano-drops technique of crystallization with a novel optimization procedure, and the structure was solved initially with the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion . The structure of Amel-ASP1 has been refined at 1.6-A resolution . Its fold is roughly similar to that of other PBP/odorant binding proteins, presenting six helices and three disulfide bridges . Contrary to the PBPs from Bombyx mori (Sandler, B . H., Nikonova, L., Leal, W . S., and Clardy, J . (2000) Chem . Biol . 7, 143-151) and Leucophea maderae (Lartigue, A., Gruez, A., Spinelli, S., Riviere, S., Brossut, R., Tegoni, M., and Cambillau, C . (2003) J . Biol . Chem . 278, 30213-30218), the extended C terminus folds into the protein and forms a wall of the internal hydrophobic cavity . Its backbone groups establish two hydrogen bonds with a serendipitous ligand, n-butyl-benzene-sulfonamide, an additive used in plastics . This mode of binding might, however, mimic that used by one of the pheromonal blend components and illustrates the binding versatility of PBPs. Ann N Y Acad Sci, 2003 Sep, 998, 539 - 48 Future therapeutic strategies in autoimmune myasthenia gravis; Psaridi-Linardaki L et al.; Antibodies against muscle acetylcholine receptor (AChR) undoubtedly play a critical role in the pathology of most myasthenia gravis (MG) cases . Selective elimination of the majority of these antibodies should result in a considerable improvement of the MG symptoms . Such a specific elimination could be achieved by AChR-based immunoadsorbents . However, sufficient quantities of native human AChR are not available while bacterially expressed recombinant domains of the AChR are unable to bind satisfactorily MG antibodies . We have undertaken the production of the extracellular domains of human AChR subunits in eukaryotic systems, in native-like conformation, for their use as potent immunoadsorbents . The N-terminal extracellular domain (amino acids 1-210; alpha(1-210)) of the alpha(1) subunit of the human muscle AChR was expressed in the yeast Pichia pastoris . The polypeptide was water-soluble, glycosylated, and in monomer form . The alpha(1-210) bound 125I-alpha-bungarotoxin (125I-alpha-BTX) with a high affinity (Kd = 5.1 +/- 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine . Several conformation-dependent anti-AChR antibodies were able to bind alpha(1-210) as did antibodies from a large proportion of MG patients . The purified protein was subsequently immobilized on Sepharose-CNBr and was used to immunoadsorb anti-AChR antibodies from 64 MG sera . It eliminated more than 50% (50-94%) of the anti-AChR antibodies in 20% of the sera, whereas from another 30% of the sera it eliminated 20-60% of their anti-AChR antibodies . Work is in progress for the expression of the extracellular domain of all other muscle AChR subunits . It is expected that their combined use may eliminate the great majority of the anti-AChR antibodies from most MG patients. Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2288 - 90 Efficient production of recombinant human pleiotrophin in yeast, Pichia pastoris; Murasugi A et al.; Approximately 260 mg/l of authentic recombinant human pleiotrophin (rhPTN) was expressed into the medium of high-cell density fermentation using a Pichia pastoris protein expression system . The prepro-sequence of yeast alpha-mating factor was used successfully . The recombinant hPTN was efficiently recovered from the medium by expanded bed adsorption, and purified using successive column chromatography steps . In the purified rhPTN preparation, modified rhPTN were scarcely detected . Circular dichroism measurement of the purified PTN showed the presence of the characteristic beta-structures in the protein. Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2277 - 9 Production of functional lectin in Pichia pastoris directed by cloned cDNA from Aleuria aurantia; Amano K et al.; A plasmid bearing a nucleotide sequence of fucose-specific lectin of Aleuria aurantia was constructed and expressed in a methylotrophic yeast, Pichia pastoris . The product showed almost the same hemagglutinating activity as the lectin produced in Escherichia coli, the properties of which were quite similar to the native one . Because of glycosylation of the product, the molecular mass was larger than that of the native one, and it acquired higher thermostability. Vaccine, 2003 Dec 1, 21(32), 4736 - 43 Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens; Pitcovski J et al.; Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry . One of the most effective types of inactivated IBDV vaccine is produced by infecting young chickens with a virulent strain, sacrificing them and extracting the virus from the bursa of Fabricius . The goal of this study was to produce an effective subunit vaccine against IBDV thereby providing an effective means of combating the disease . In areas in which the bursa-derived vaccine is in use, this subunit vaccine would eliminate the use of live birds for the production of inactivated vaccines . The gene for viral protein 2 (VP2) of IBDV was cloned into a Pichia pastoris expression system . This efficient system allowed us to meet the need for inexpensive vaccines required by the poultry industry . Following expression and scale-up, the protein was used to vaccinate chickens, against either Gumboro disease alone or in combination with inactivated Newcastle disease virus (NDV) . Full protection was conferred against IBDV following vaccination with the subunit recombinant vaccine . No untoward influence on the response to the NDV vaccine was recorded . Over 250 million birds have already been vaccinated with this vaccine . The advantages of a subunit vaccine over an inactivated one are discussed . This approach will enable rapid adjustment to new virulent strains if and when they appear. Cell Biol Int, 2003, 27(11), 947 - 52 Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica; Stasyk OV et al.; Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared . These mutants do not contain the specific ergosterol derivate, ergosterol glucoside . We observed that the P . pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol . PpUGT51 is also required for other vacuole related processes . In contrast, the Y . lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy . Thus, sterol glucosyltransferases play different functional roles in P . pastoris and Y . lipolytica. Arch Virol, 2003 Nov, 148(11), 2267 - 73 Carboxy-terminally truncated Dengue 4 virus envelope glycoprotein expressed in Pichia pastoris induced neutralizing antibodies and resistance to Dengue 4 virus challenge in mice; Mune M et al.; We have expressed a recombinant Dengue 4 virus envelope glycoprotein (E4rec), truncated at its C-terminus by 53 amino acids, in Pichia pastoris . The presence of E4rec was confirmed by Western-blot using anti-DEN 4 hyper immune mouse ascitic fluid . E4rec migrated during SDS-PAGE as a 64 kDa protein . Treatment with endoglycosidases showed that the E protein was modified by the addition of short mannose chains and the absence of hyperglycosylation . When administered to BALB-C mice, E4rec elicited a DEN 4 neutralizing antibody response haemagglutination inhibition antibodies and specific memory T cell response . Mice immunized were also significantly protected against lethal DEN 4 virus challenge (86.6%, p < 0.001). Trends Biotechnol, 2003 Nov, 21(11), 459 - 62 Genetic engineering of Pichia pastoris to humanize N-glycosylation of proteins; Bretthauer RK; The yeast Pichia pastoris is used extensively as the host cell for large-scale production of secreted recombinant proteins . Many proteins of pharmaceutical importance are N-glycosylated, and therefore require an expression host that yields N-linked oligosaccharides that are structurally and functionally identical to the human counterpart . The recent report by Choi et al . describes the use of combinatorial genetic libraries to alter the N-glycosylation pathway in P . pastoris to yield N-linked oligosaccharides with hybrid structures that are the same as the intermediates of mammalian-protein N-glycosylation . In view of recent progress in this area, the production of complex human glycans in yeasts is anticipated. FEBS Lett, 2003 Oct 23, 553(3), 365 - 9 Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris; Hillig I et al.; In plants, glucosylceramide (GlcCer) biosynthesis is poorly understood . Previous investigations suggested that sterol glucoside (SG) acts as the actual glucose donor for the plant GlcCer synthase (GCS) . We addressed this question by generating a Pichia pastoris double mutant devoid of GlcCer and SG . This mutant was used for heterologous expression of the plant GCS . The activity of the GCS resulted in the accumulation of GlcCer and, surprisingly, a small proportion of SG . The synthesis of GlcCer in the transformed double mutant shows that the GCS is SG-independent, while the detection of SG suggests that in addition to the sterol glucosyltransferase, also the GCS may contribute in planta to SG biosynthesis. Biotechnol Lett, 2003 Sep, 25(18), 1525 - 30 A cDNA encoding vacuolar type beta-D-fructofuranosidase (Os beta fruct3) of rice and its expression in Pichia pastoris; Fu RH et al.; A vacuolar type beta-D-fructofuranosidase (Os beta fruct3) was cloned from etiolated rice seedlings cDNA library . It encodes an open reading frame of 688 residues . The deduced amino acid sequence had 58% identity to the vacuolar type beta-D-fructofuranosidase of maize (Ivr1) . Os beta fruct3 exists as a single copy per genome . Northern analyses showed that Os beta fruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars . Os beta fruct3 was also heterologously expressed in Pichia pastoris . The recombinant proteins were confirmed to be a vacuolar type beta-D-fructofuranosidase. Yeast, 2003 Oct 15, 20(13), 1097 - 108 The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter; Menendez J et al.; We cloned and characterized a gene encoding isocitrate lyase from the methylotrophic yeast Pichia pastoris . This gene was isolated from a P . pastoris genomic library using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed from conserved regions in yeast isocitrate lyases . The cloned gene was sequenced and consists of an open reading frame of 1563 bp encoding a protein of 551 amino acids . The molecular mass of the protein is calculated to be 60.6 kDa with high sequence similarity to isocitrate lyase from other organisms . There is a 64% identity between amino acid sequences of P . pastoris Icl and Saccharomyces cerevisiae Icl . Northern blot analyses showed that, as in S . cerevisiae, the steady-state ICL1 mRNA levels depend on the carbon source used for cell growth . Expression in P . pastoris of the dextranase gene (dexA) from Penicillium minioluteum under control of the ICL1 promoter proved that P(ICL1) is a good alternative for the expression of heterologous proteins in this methylotrophic yeast . The sequence presented here has been deposited in the EMBL data library under Accession No . AJ272040 . Protein Expr Purif, 2003 Oct, 31(2), 213 - 21 Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp; Aoki H et al.; A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris . We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene . In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography . The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached . The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture . The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L . The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes. Protein Expr Purif, 2003 Oct, 31(2), 197 - 206 Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization; Li A et al.; Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury . We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa . The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies . Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H . We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation . Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases . Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography . The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification . Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies. Protein Expr Purif, 2003 Oct, 31(2), 173 - 80 Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris; Grzelak K et al.; Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da . This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris) . The expression in bacteria was low and the protein was rapidly degraded upon cell lysis . The expression of His8-tagged rJHBP (His8-rJHBP) in P . pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography . His8-rJHBP from P . pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M . The results suggest that P . pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form. Antonie Van Leeuwenhoek, 2003, 84(2), 147 - 59 Six new methanol assimilating yeast species from wood material; Peter G et al.; Ten yeast strains representing six hitherto unknown methanol utilizing yeast species were isolated from tree exudate, bark and rotten wood samples . Following the sequencing of the D1/D2 region of their large (26S) subunit rDNA, the four ascosporogenous species were assigned to the genus Pichia, and the two anascosporogenous ones to the genus Candida . Although genetically clearly separated, three of the four new Pichia species are phenotypically very similar to P . pini, and they can be differentiated only by minor physiological and morphological characteristics . The description is given for the six new species (C . suzukii, C . hungarica, P . trehaloabstinens, P . pilisensis, P . dorogensis and P . zsoltii). Appl Environ Microbiol, 2003 Oct, 69(10), 6064 - 72 Optimized expression of a thermostable xylanase from Thermomyces lanuginosus in Pichia pastoris; Damaso MC et al.; Highly efficient production of a Thermomyces lanuginosus IOC-4145 beta-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter . P . pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures . Furthermore, an optimization of enzyme production was carried out with a 2(3) factorial design . The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter . A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved . Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior . Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus . The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical . For both enzymes, the optimum temperature was 75 degrees C, and they retained 60% of their original activity after 80 min at 70 degrees C or 40 min at 80 degrees C . The high level of fully active recombinant xylanase obtained in P . pastoris makes this expression system attractive for fermentor growth and industrial applications. Biotechnol Prog, 2003 Sep-Oct, 19(5), 1403 - 9 Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris; Gu L et al.; A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris . Three different expression vectors were constructed: pZBMNP contains the native P . chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression . Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P . pastoris transformants . The majority of the rMnP produced by P . pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa) . Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P . pastoris . Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation . Partially purified rMnP showed kinetic characteristics similar to those of wtMnP . Both enzymes also had similar pH stability profiles . Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes . However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C. Microbiology, 2003 Oct, 149(Pt 10), 2931 - 9 Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product; Jaques AK et al.; The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1 . Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action . That is, the beta-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases . Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc(4), GlcNAc(5) and GlcNAc(6) indicated that the predominant reaction involved hydrolysis of GlcNAc(2) from the non-reducing end of each substrate . Products of transglycosylation were also identified in each incubation . Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical . However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type . The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A . fumigatus TIGR database . J Mol Biol, 2003 Oct 10, 333(1), 103 - 16 Structural characterisation of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris; Parcej DN et al.; Neuronal voltage-dependent K(+) channels of the delayed rectifier type consist of multiple Kv alpha subunit variants, which assemble as hetero- or homotetramers, together with four Kv beta auxiliary subunits . Direct structural information on these proteins has not been forthcoming due to the difficulty in isolating the native K(+) channels . We have overexpressed the subunit genes in the yeast Pichia pastoris . The Kv1.2 subunit expressed alone is shown to fold into a native conformation as determined by high-affinity binding of 125I-labelled alpha-dendrotoxin, while co-expressed Kv1.2 and Kv beta 2 subunits co-assembled to form native-like oligomers . Sites of post-translational modifications causing apparent heterogeneity on SDS-PAGE were identified by site-directed mutagenesis . Engineering to include affinity tags and scale-up of production by fermentation allowed routine purification of milligram quantities of homo- and heteroligomeric channels . Single-particle electron microscopy of the purified channels was used to generate a 3D volume to 2.1 nm resolution . Protein domains were assigned by fitting crystal structures of related bacterial proteins . Addition of exogenous lipid followed by detergent dialysis produced well-ordered 2D crystals that exhibited mostly p12(1) symmetry . Projection maps of negatively stained crystals show the constituent molecules to be 4-fold symmetric, as expected for the octameric K(+) channel complex. Genetika, 2003 Aug, 39(8), 1026 - 32 {The red mutations impair the regulation of flavinogenesis and metal homeostas in yeast Pichia guilliermondii}; Stenchuk NN et al.; A method of positive selection of mutants with impaired regulation of flavinogenesis and metal homeostasis in yeast Pichia guilliermondii was developed . This positive selection system was based on the isolation of pseudo-wild-type revertants (the Rib+ phenotype) in riboflavin-dependent rib1-86 mutant (the Rib- phenotype) of yeast P . guilliermondii . Mutation rib1-86 blocks activity of the GTP cyclohydrolase II catalyzing the first step in riboflavin (RF) biosynthesis . Study of a collection of spontaneous Rib+ revertants allowed the identification of a considerably large number of genetic loci responsible for the suppression of rib1-86, which include both previously identified three loci (rib80, rib81, and hit1) and six new loci designated red1-red6 (reduction) . A comparative analysis of the wild-type strain and red mutants revealed that these mutants had higher activity levels of GTP cyclohydrolase and RF-synthase, elevated levels of RF biosynthesis, enhanced Fe/Cu reductase activity and higher total iron content in cells and that they are characterized by enhanced sensitivity to transition metals (Fe(III), Cu(II), Cd(II), Co(II), Zn(II), Ag(I), and to H2O2 . The metal hypersensitivity of mutant cells can be prevented by an increased amount of extracellular iron ions . Mutations red1 and red6 synergistically interact with the locus rib81 in the course of RF biosynthesis . Obviously, each RED gene plays an important role in the regulation of both flavinogenesis and metal homeostasis in P . guilliermondii cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Oct, 35(10), 941 - 6 Isolation of a multi-functional endogenous cellulase gene from mollusc, Ampullaria crossean; Wang J et al.; The cellulase genes of some animals, most coding for endo-beta-1,4-glucanases, were found and cloned . There has been no reports about genes encoding exo-beta-1,4-glucanase or endo- -1,4-xylanase from animal . Here we cloned the cDNA of a cellulase designated as EGX from mollusc, Ampullaria crossean, and expressed it in Pichia pastoris for the first time . The cellulase EGX is a multi-functional beta cellulase with the activities of exo-beta-1,4-glucanase, endo-beta-1,4-glucanase and endo-beta-1,4-xylanase . The opening reading frame of EGX cDNA is 1185 bp and encodes 395 amino acids . The EGX gene can also be amplificated from the genomic DNA by PCR, which verified the endogenous origin of this gene . This EGX gene was the first multi-functional cellulase gene that was directly isolated from animals. Glycobiology, 2004 Jan, 14(1), 27 - 37 Epub 2003 Sep 26. Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein; Medzihradszky KF et al.; MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits . After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor . MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris . Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios . To test this, glycosylated MFE-CP was expressed and purified from P . pastoris . The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo . Furthermore, it showed effective tumor localization . This favorable glycosylation pattern was analyzed by tandem mass spectrometry . High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task . Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures . Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length . Asn-484 was found only in its nonglycosylated form . There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units . Nonglycosylated forms of Asn-442 and Asn-492 were not observed. Biotechnol Lett, 2003 Aug, 25(15), 1281 - 5 Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris; Baumgartner P et al.; The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris . Transformants of P . pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed . GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography . The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity. Biotechnol Lett, 2003 Sep, 25(17), 1457 - 62 Expression of the gene coding for bacterial hemoglobin improves beta-galactosidase production in a recombinant Pichia pastoris; Wu JM et al.; Genes coding for Vitreoscilla hemoglobin (VHb) with peroxisome targeting signal (PTS1) tag and beta-galactosidase were co-expressed in Pichia pastoris under the alcohol oxidase1 (AOX1) promoter . The expression of VHb-PTS1 had no positive effect on cell growth but significantly enhanced the whole cell beta-galactosidase activity to 4-fold higher than that of VHb-free cell in yeast extract/peptone/methanol medium under aerobic cultivation. Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 54 - 8 Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient; Falcon V et al.; Little is known about the life cycle of hepatitis C virus . Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins . HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems . In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient . They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells . In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy . This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection. Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 48 - 53 Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast; Acosta-Rivero N et al.; The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied . At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus . HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus . However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm . This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P . pastoris yeast. J Interferon Cytokine Res, 2003 Jul, 23(7), 351 - 8 Generation and characterization of recombinant unmodified and phosphorylatable murine IFN-alpha1 in the methylotropic yeast Pichia pastoris; Trajanovska S et al.; In order to generate reagents to study the murine type I interferon (IFN) system, recombinant murine IFN-alpha1 (rMuIFN-alpha1) protein was expressed in the methylotropic yeast Pichia pastoris . rMuIFN-alpha1 with a phosphate acceptor site engineered at the C-terminus (rMuIFN-alpha1P) to enable radiolabeling by gamma(32)P-ATP and cAMP-dependent protein kinase was also generated . Proteins of 20, 25 (MuIFN-alpha1) and 25.5 (MuIFN-alpha1P), kDa were detected in the yeast growth medium, had type I IFN activity, and were recognized by antimurine L929 cell IFN antibodies . The MuIFN-alpha1 proteins produced in P . pastoris were a mixture of glycosylated and unglycosylated forms, with sugars of approximately 5 kDa added via N-linked glycosylation . The recombinant proteins were highly purified using a single RP-HPLC elution step, and their authenticity was confirmed by amino-terminal amino acid sequencing . The MuIFN-alpha1 and MuIFN-alpha1P protein preparations had specific antiviral activities of 1.3 x 10(7) and 4.7 x 10(6) IU/mg protein, respectively . MuIFN-alpha1P could be radiolabeled to a high specific radioactivity (0.6-2 x 10(8) cpm/microg protein) with gamma(32)P-ATP and cAMP-dependent protein kinase without significantly altering its biologic activity or electrophoretic properties . Binding experiments on COS-7 cells transiently transfected with MuIFNAR-2 and IFNAR-2 demonstrated specific and dose-dependent binding of gamma(32)P-ATP-MuIFN-alpha1P to cell surface type I IFN receptors. Virus Genes, 2003 Oct, 27(2), 189 - 96 High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Pichia pastoris; Qian P et al.; High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult . In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115 . Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5'-end mRNA of foreign gene . The modified gene was inserted into the yeast expression vector pPICZalphaA, induced and expressed by the same methods . The recombinant protein with a molecular mass of approximately 23 kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV . The results indicated that it was similar to the native protein . The expression level of the recombinant protein could attain 2.0 g/L . In the meanwhile, the optimal conditions for expression were determined . It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene. Appl Microbiol Biotechnol, 2003 Dec, 63(2), 153 - 8 Epub 2003 Sep 16. Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii; Colao MCh et al.; A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201 . This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide . Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity) . The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium . The level of lcc1 transcription was higher when T . trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone . The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism . The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase. Mol Biol Cell, 2004 Jan, 15(1), 58 - 70 Epub 2003 Sep 17. Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure; Mukaiyama H et al.; Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation . Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown . In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae . We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole . Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system . We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation. Am J Trop Med Hyg, 2003 Aug, 69(2), 129 - 34 Induction of neutralizing antibodies and partial protection from viral challenge in Macaca fascicularis immunized with recombinant dengue 4 virus envelope glycoprotein expressed in Pichia pastoris; Guzman MG et al.; A recombinant vaccine that expresses the envelope (E) gene of dengue virus type 4 was tested for immunogenicity and protection in Macaca fascicularis . One hundred micrograms of semipurified recombinant E protein (E4rec) expressed in Pichia pastoris was used to immunize three animals . Neutralizing antibodies to dengue 4 virus with a titer of 1:30 were detected in all immunized monkeys prior to challenge . Animals were challenged with 10(5) plaque-forming units of dengue 4 virus . One vaccine-immunized monkey was protected from viremia, while the other two were partially protected . Monkeys immunized with E4rec elicited the highest neutralizing antibody titers (P < 0.05) ranging from 1:85 to 1:640 at day 30 . In both immunized and control animals, the longest duration of viremia correlated with earliest and highest level of IgM antibody to dengue virus . The vaccinated animals showed anamnestic antibody responses upon virus challenge, indicating successful priming by the recombinant vaccine . Our results suggest that E4rec expressed in P . pastoris can provide partial protection against viremia . However, the results were not effective enough to use it as a vaccine candidate . Further work is required to improve the quality of the immunogen. Di Yi Jun Yi Da Xue Xue Bao, 2003 Sep, 23(9), 895 - 8 {Expression of recombinant human endostatin in Pichia pastoris and its inhibitory effects on the growth of human lungadenocarcinoma Astc-a-1 cells}; Zhang L et al.; OBJECTIVE: To achieve expression of recombinant human endostatin (rhES) in a highly efficient foreign gene expression system, the yeast strain Pichia pastoris, and to evaluate the inhibitory effect of rhES on the growth of nude mouse pulmonary adenocarcinoma cells . METHODS AND RESULTS: The rhES gene was efficiently expressed in the yeast strain, and heparin affinity chromatography yielded highly purified endostatin identified by Western blotting, which proved to significantly inhibit the growth of the ECV-304 cells in vitro and also the growth of the lung adenocarcinoma Astc-a-1 in nude mice . CONCLUSIONS: The rhES gene can be expressed in Pichia pastoris, and has the ability to restrain the growth of ECV-304 cells and lung adenocarcinoma Astc-a-1 in nude mice, showing important potentials for future clinical applications. Mol Biochem Parasitol, 2003 Sep, 131(1), 65 - 75 Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase; Sajid M et al.; Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni . This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition . Using irreversible affinity probes, we demonstrate that S . mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts . SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)) . Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms . Recombinant SmCB1pm was unable to catalyze its activation, even at low pH . However, recombinant S . mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans . Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence . The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo . The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles . Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded . Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu) . This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo. Protein Expr Purif, 2003 Sep, 31(1), 115 - 22 Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease; Cabral KM et al.; Plant defensins are small cysteine-rich proteins that present high activity against fungi and bacteria and inhibition of insect proteases and alpha-amylases . Here, we present the expression in Pichia pastoris, purification and characterization of the recombinant Pisum sativum defensin 1(rPsd1); a pea defensin which presents four disulfide bridges and high antifungal activity . For this, we had to overcome the inefficiency of the STE13 protease . Our strategy was to clone the corresponding cDNA directly in-frame with a variant of the widely used secretion signal from the Saccharomyces cerevisiae alpha-mating factor, devoid of the STE13 proteolytic signal cleavage sequence . Using an optimized expression protocol, which included a buffered basal salt media formulation, it was possible to obtain about 63.0mg/L of 15N-labeled and unlabeled rPsd1 . The recombinants were purified to homogeneity by gel filtration chromatography, followed by reversed-phase HPLC . Mass spectrometry of native and recombinant Psd1 revealed that the protein expressed heterologously was post-translationally processed to the same mature protein as the native one . Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein had the same folding when compared to native Psd1 . In addition, the rPsd1 was fully active against Aspergillus niger, if compared with native Psd1 . To our knowledge, this is the first heterologous expression of a fully active plant defensin in a high-yield flask. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 643 - 51 Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris; Reddy ST et al.; A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene . The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography . Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase . To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man . The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site . Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain . In conclusion, the full-length CD-MPR produced in P . pastoris is structurally and functionally suitable for crystallization studies. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 637 - 42 A novel disintegrin-like domain of a high molecular weight metalloprotease inhibits platelet aggregation; You WK et al.; Disintegrin is one