Mutat Res, 1996 Oct 25, 357(1-2), 67 - 74 Radiosensitivity of haplont yeast cells irradiated with sparsely and densely ionizing radiations; Korogodin VI et al.; Five haploid and three diploid yeast strains of various species (Yarrowia lipolytica, Pichia pinus and Pichia guilliermondii) were irradiated with alpha-particles from 239Pu and gamma-rays from 137Cs or 60Co in the stationary phase of growth . A common feature of these species is that they exhibit a haploid state as a normal vegetative state in natural conditions . It was shown that the transition from the haploid to the diploid state is not accompanied by increased radioresistance, and diploid strains were unable to perform liquid-holding recovery . The absence of diploid-specific recovery in diploid strains was also supported by the fact that the RBE of alpha-particles was almost identical for haploid and the corresponding diploid strains being much smaller than that observed in typical wild-type diploid strains capable of diploid-specific recovery . The results suggest that haplont yeast may have evolved to diplont yeast via the development of a specific repair system conferring specific resistance in the diploid state. Gene, 1996 Oct 24, 177(1-2), 163 - 7 Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris; Raschke WC et al.; Pichia pastoris (Pp) and Hansenula polymorpha (Hp) are methylotrophic yeasts commonly used for industrial purposes . Growth of either of these yeasts in the presence of methanol as the carbon source results in high-level induction of alcohol oxidase expression . The respective alcohol oxidase genes, AOX1 in Pp and MOX in Hp, have similar regulatory characteristics . Our studies show that the Pp AOX1 promoter (AOX1p) can be used for methanol-induced expression of a heterologous gene in Hp . Furthermore, the size of an AOX1p-heterologous gene-AOX1 terminator cassette transcript synthesized in Hp is indistinguishable from that synthesized in Pp suggesting that transcription both initiates and terminates at the same sites in both yeast species . Induction of AOX1p in Hp demonstrates that the methanol-inducible regulatory mechanism in Hp is able to recognize and activate the Pp promoter in spite of extensive sequence variations between AOX1p and MOXp. Gene, 1996 Oct 24, 177(1-2), 69 - 76 Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris; Okabayashi K et al.; To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV) . The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris . Transformants were monitored for the secretion of fibrinolytic activity . The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions . It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence . Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis . Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis . Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium . The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin . The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation . Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV. Carbohydr Res, 1996 Oct 23, 293(1), 101 - 17 The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the structure of the phosphomannan backbone; Parolis LA et al.; The phosphomannan core of the exopolysaccharide of Pichia (Hansenula) holstii NRRL Y-2448 was isolated after hydrolytic removal of the oligosaccharide phosphate side-chains . The core polysaccharide and its dephosphorylated derivative were subjected to extensive 1D and 2D NMR spectroscopy which yielded information on the linkage sites and on the sequence of the mannosyl residues in the major oligosaccharide repeating unit . The most probable structure for the repeating unit was -{6-O-PO3H2-alpha-D-Man-(1-->3)-alpha-D-Man-(1-->2)-alpha-D-Man-(1 -->2)}-alpha-D-Man-(1-->6)-{alpha-D-Man-(1-->2)}-alpha-D-Man-(1-->6)- . A semiquantitative conformational analysis was performed by Monte Carlo simulations and the result was confirmed by comparison with the experimentally determined NMR data . The distance distribution for the phosphate groups was determined from the modeling and was found to cover the expected range of distances for phosphorylated high-mannose oligosaccharides. Gene, 1996 Oct 17, 176(1-2), 197 - 201 Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris; Battista MC et al.; We have previously shown that single or multiple epitopes of the major human cytomegalovirus (HCMV) antigens, produced as fusion proteins in prokaryotes can be valuable diagnostic material in the serology of HCMV infection . In this work we moved to a eukaryotic system, to produce one of the most immunogenic HCMV antigens, ppUL44 (also called pp52 due to its apparent molecular size on acrylamide gels), as a non-fusion protein, in an attempt to eliminate some non-specific reactivity of human sera with bacterial carrier proteins . We expressed the DNA encoding ppUL44 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris . Good levels of intracellular, soluble pp52 were produced . We observed an indistinguishable pattern of the yeast pp52 from the viral native protein in immunoblotting and a good reactivity with human sera. FEBS Lett, 1996 Oct 7, 394(3), 268 - 72 Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris; Talmont F et al.; The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene . Cell membranes exhibited specific and saturable binding of the opioid antagonist {3H}diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell) . Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes. Curr Opin Biotechnol, 1996 Oct, 7(5), 525 - 30 Quality and authenticity of heterologous proteins synthesized in yeast; Eckart MR et al.; Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments . There has been recent progress in characterizing and controlling the factors involved in determining authenticity. Curr Opin Biotechnol, 1996 Oct, 7(5), 517 - 24 The expression of recombinant proteins in yeasts; Sudbery PE; The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris are rapidly becoming the systems of choice for the expression of recombinant proteins in yeast . However, the powerful genetic techniques available in Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are still exploited to establish models to study medically important cell processes and screen for pharmacologically active compounds. Appl Environ Microbiol, 1996 Oct, 62(10), 3894 - 6 A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene; Liden G et al.; The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2 . The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink . The specific xylitol production rate obtained was 0.38 g g-1 h-1. Appl Environ Microbiol, 1996 Oct, 62(10), 3864 - 7 Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii; Pagot Y et al.; This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii . The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate . The subcellular sites and induction patterns were studied . The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo . We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates. Curr Microbiol, 1996 Oct, 33(4), 237 - 42 Characterization of the Genetic System of the Xylose-Fermenting Yeast Pichia stipitis Melake T, Passoth V V, Klinner U. High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid . Sporulation ability of these clones pointed to a homothallic life cycle . Mating was induced by cultivation under nutritionally poor conditions on malt extract medium . Conjugation was followed immediately by sporulation . However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started . Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate . The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Yeast, 1996 Sep 30, 12(12), 1187 - 200 Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris; Roca H et al.; The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe . Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns . Amino acid sequences comparison of P . minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp . CB-8 . The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter . Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol . The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical. Biochim Biophys Acta, 1996 Sep 13, 1297(1), 99 - 104 Trp-16 is essential for the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase; Zhu A et al.; By expressing site-directed mutants in the methylotrophic yeast strain Pichia pastoris, the role of a tryptophan residue at position 16 in the activity of alpha-galactosidase and alpha-N-acetylgalactosaminidase, two closely related exoglycosidases, was studied . A substitution of Trp-16 with an arginine residue in alpha-N-acetylgalactosaminidase abolished the enzyme activity, which was confirmed by replacing a 600 bp fragment containing the mutation with the corresponding wild-type sequence . The same tryptophan residue was then substituted with an alanine in both enzymes by site-directed mutagenesis to reveal a possible relationship between their active sites . The purified alpha-N-acetylgalactosaminidase mutant demonstrated a specific activity of 2.8 x 10(-2) U/mg and a Vmax/K(m) of 4.3 x 10(-2), which were both more than a thousandfold lower than corresponding values for the wild-type enzyme . Furthermore, the mutant failed to bind to an affinity resin, suggesting the involvement of Trp-16 in substrate-binding . In addition, the purified alpha-galactosidase mutant resulted in more than a 10(4)-fold decrease in specific activity . Thus our data suggest that Trp-16 in both alpha-galactosidase and alpha-N-acetylgalactosaminidase is critical for enzymatic activity, which in turn supports the hypothesis that these two enzymes may share a catalytic mechanism involving similar residues in their active sites. Plant Cell Physiol, 1996 Sep, 37(6), 816 - 24 Isolation and characterization of mitochondrial nucleoids from the yeast Pichia jadinii; Miyakawa I et al.; Mitochondrial (mt) nucleoids were isolated with a high degree of purity from the yeast Pichia jadinii, in which the mitochondrial DNA (mtDNA) is linear . Field-inversion gel electrophoresis (FIGE) revealed that significant amounts of mtDNA could be isolated intact, as linear molecules of 41 kbp, from the isolated mt-nucleoids . Fifteen different proteins were detected in the mt-nucleoid fraction and, eight of these proteins bound to DNA . The patterns of mt-nucleoid proteins and of the DNA-binding proteins after gel electrophoresis in the presence of SDS were somewhat different from those of such proteins from Saccharomyces cerevisiae . The corresponding proteins isolated from the mt-nucleoids of four other species of yeast in the genera Pichia and Williopsis also differed from one another in terms of electrophoretic mobility in the presence of SDS . In immunoblotting experiments, antibodies that had been raised against the 67-kDa protein of mt-nucleoids from S.cerevisiae and the YMN-1 monoclonal antibody that is specific for a 48-kDa protein in the mt-nucleoids from S . cerevisiae did not recognize any proteins in the mt-nucleoids from Pichia jadinii and four other species of yeast . The results suggest the considerable diversity of the proteins in the mt-nucleoids of yeasts. Protein Expr Purif, 1996 Sep, 8(2), 254 - 61 Overexpression in Pichia pastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthora cryptogea; O'Donohue MJ et al.; A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris . Having first optimized the gene construction for secretion, we have overexpressed a modified beta-cryptogein in a secreted form . A purification scheme suited to this expression system has been developed and highly pure, biologically active protein has been obtained . For structural analysis of this recombinant beta-cryptogein, and new mutated forms thereof, optimal conditions for the crystallization of this protein have been determined and crystals that diffract to 2.2 A have been obtained. Protein Expr Purif, 1996 Sep, 8(2), 204 - 14 Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris; Juge N et al.; Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae . The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2 . Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose . The N-terminal sequence, pI, and Mr indicated that native-like processing took place . Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1 . While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS . The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity . Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts . This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships. J Gen Virol, 1996 Sep, 77 ( Pt 9), 2001 - 8 Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus; Eckhart L et al.; In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones . Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus . We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg) . Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles . Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans . More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed . Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro . These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented . Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component. FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 147 - 53 Isolation and characterisation of mutants from the halotolerant yeast Pichia sorbitophila defective in H+/glycerol symport activity; Oliveira RP et al.; Pichia sorbitophila, a yeast species that is highly resistant to osmotic stress in general and to salt stress in particular, was subjected to a mutagenesis strategy in order to obtain mutants deficient in the glycerol active uptake previously described . Density centrifugation was used for enrichment of NaCl sensitive mutants in either glucose or glycerol media . Several phenotypic classes of mutants were identified, to which physiological tests were applied concerning the activity of the symporter, its accumulation capacity and the detection of the activity of glycerol pathway specific enzymes . From these, two mutant strains were selected, presenting a clearly deficient phenotype on H+/glycerol symport activity. J Biol Chem, 1996 Aug 23, 271(34), 20594 - 602 Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris . Calmodulin response is complete within the reductase domain itself; Gachhui R et al.; Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain . Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c . To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response . The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture . It contained 1 FAD and 0.8 FMN per molecule . Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation . Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH . Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS . Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence . We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself. Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8989 - 94 The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome; Wu SH et al.; Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins . Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group . Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein . The hypothesis that P . pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study . Soluble extracts from P . pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A . HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin . These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor. J Biol Chem, 1996 Aug 16, 271(33), 20156 - 62 Expression of recombinant HLA-DR2 molecules . Replacement of the hydrophobic transmembrane region by a leucine zipper dimerization motif allows the assembly and secretion of soluble DR alpha beta heterodimers; Kalandadze A et al.; Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells . Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/peptide/T cell receptor recognition unit . The alpha and beta chains of DR2 (encoded by the DRA, DRB1*1501 genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated . The hydrophobic transmembrane regions of DRalpha and DRbeta facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure . The DRalpha-Fos and DRbeta-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the alpha-mating factor secretion signal to direct expression to the secretory pathway . DR alphabeta heterodimers were purified from supernatants using an antibody specific for the DR alphabeta heterodimer . Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide . Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2.peptide complexes. Arch Biochem Biophys, 1996 Aug 15, 332(2), 305 - 12 Identification of a novel pelD gene expressed uniquely in planta by Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of its protein product as an endo-pectate lyase; Guo W et al.; Antibodies prepared against a pectin-inducible pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) were previously found to protect the host from infection . The gene (pelA) and two of its homologs were cloned and sequenced . Here we report the isolation of a new pectate lyase gene, pelD, from a genomic library of F . solani pisi . A 1.5-kb DNA fragment containing pelD and its flanking regions was sequenced . The nucleotide sequence of pelD would encode a protein of 24.5 kDa which shares 49, 44, and 65% amino acid sequence identity with PLA, PLB, and PLC, respectively, from the same fungus . Because the first 19 amino acid residues appeared to be a signal peptide, the mature enzyme could be a 22.7-kDa protein . pelD transcripts and PLD protein could not be detected in fungus cultured in glucose, pectin, pea epicotyl extract, or a pea cell wall preparation . However, pelD transcripts were readily found by RT-PCR with RNA isolated from infected pea tissues . The cDNA of pelD, thus obtained, was expressed in Pichia pastoris with the putative pelD signal sequence . The secreted PLD was purified and characterized to be an endopectate lyase, and its lyase activity could be inhibited by anti-PLA IgG . Thus, protection of the host observed with the anti-PLA antibodies could reflect inhibition of immunologically related pectate lyases including PLD which is expressed uniquely in planta. Biochem J, 1996 Aug 15, 318 ( Pt 1), 125 - 31 Drosophila melanogaster angiotensin I-converting enzyme expressed in Pichia pastoris resembles the C domain of the mammalian homologue and does not require glycosylation for secretion and enzymic activity; Williams TA et al.; Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains) . In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris . The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains . Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant . Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO) . This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE . However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions . Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis . A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites. J Biol Chem, 1996 Aug 2, 271(31), 18973 - 80 Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris; Wiemer EA et al.; The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis . We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa . In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy . No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis . PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein . Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane . PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p . The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly . A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences . The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed. J Biol Chem, 1996 Aug 2, 271(31), 18310 - 3 Synthesis and cleavage- secretion of enzymatically active rabbit angiotensin-converting enzyme in Pichia pastoris; Sadhukhan R et al.; Many biologically important ectoproteins that are anchored in the plasma membrane via a hydrophobic domain undergo a proteolytic cleavage process, which releases the ectodomain to the extracellular milieu in a regulated fashion . Angiotensin-converting enzyme (ACE) is one such protein that is secreted from human and mouse cells by its cleavage at one of two alternative sites in the ectodomain . Here, we report similar cleavage-secretion of ACE in the yeast Pichia pastoris . The cleavage site used in yeasts was identical to one of the two sites used in mouse cells . Moreover, as in mammalian cells, ACE secretion in yeast was inhibited by compound 3, a potent inhibitor of the metzincin family of metalloproteases . ACE proteins cleavage-secreted from yeast and from mammalian cells had identical enzymatic properties . These results demonstrate the existence of a secretase activity in yeast whose properties closely resemble those of the mammalian ACE secretase. Lett Appl Microbiol, 1996 Aug, 23(2), 79 - 84 Fatty acid patterns of film-forming yeasts and new evidence for the heterogeneity of Pichia membranaefaciens; Noronha-da-Costa P et al.; Total fatty acids (C14:0 to C18:3) of 50 strains assigned by classical identification methods to Pichia membranaefaciens (28), P . anomala (15), Dekkera anomala (2), D . bruxellensis (2) and Candida vini (3) were determined and data analysed by multivariate statistical procedures . Principal components cluster analysis defined six groups of strains . Thirteen strains of P . anomala formed a well-defined cluster, whereas P . membranaefaciens was split into three groups . The taxonomic status of the P . membranaefaciens strains was evaluated by determination of the G + C content and DNA-DNA hybridization . The results provided evidence in support of P . membranaefaciens encompassing distinct species . Only eight strains were certified as P . membranaefaciens and six were included in the same cluster, indicating that numerical analysis of fatty acid profiles points to genetic differences which remain undetectable by conventional phenotypic tests. J Biochem (Tokyo), 1996 Aug, 120(2), 229 - 32 Construction and expression of bi-functional proteins of single-chain Fv with effector domains; Luo D et al.; We fused various polypeptide extensions to the C-termini of single chain Fv (scFv) and disulfide-stabilized Fv (dsFv) fragments to facilitate detection of bi-functional proteins or to add biological effector domains, which included the human metallothionein (HMT) motif and biotin mimetic sequence . These bi-functional proteins were expressed and secreted in a recombinant Pichia pastoris system and showed specific anti-idiotype binding activity, as determined by competitive radioimmunoassaying . However, the fusion protein constructed with dsFv- HMT, but not scFv-HMT, had lost this binding activity . The interruption of the structural conformation as a result in dsFv-HMT may be explained by the interactions between the cysteines engineered in dsFv domains and the cysteines in the HMT region. Yakugaku Zasshi, 1996 Aug, 116(8), 622 - 9 {Ligand binding properties and esterase-like activity of recombinant human serum albumin}; Takeshima K et al.; The ligand binding properties and esterase-like activity of recombinant human serum albumin (rHSA) expressed by Pichia pastoris were compared with those of plasma derived human albumin (pHSA) . The binding of long fatty acid ions was determined by the equilibrium partition method using radiolabeled palmitate . The association constants and the number of binding sites of diazepam, salicylate and warfarin were determined by specific and nonspecific binding models . The high affinity binding of bilirubin was kinetically determined from the oxidation rate of free bililubin in the binding mixture . The binding parameters of these five ligands obtained with rHSA were within the same range observed with pHSA preparations . The kinetic parameters for hydrolytic activity of rHSA toward p-nitrophenyl acetate was also similar to pHSA . These results indicate that rHSA and pHSA have the same functional property. Protein Expr Purif, 1996 Aug, 8(1), 119 - 25 Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris; Mason AB et al.; The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter . Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter . As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system . Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues. FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 227 - 31 Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose; Amador P et al.; The mutant IGC 40 x 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior . Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced . This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity . Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain . In P . anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions. J Allergy Clin Immunol, 1996 Aug, 98(2), 331 - 43 Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen; Smith PM et al.; BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy . OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris . METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification . Recombinant Cyn d 1 was expressed in Escherichia coli and yeast . The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen . RESULTS: Two isoforms of Cyn d 1 were cloned . Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen . Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE . CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen. Appl Environ Microbiol, 1996 Aug, 62(8), 2832 - 8 Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of glucose and xylose metabolism in cell suspensions and agarose-immobilized cultures of Pichia stipitis and Saccharomyces cerevisiae; Lohmeier-Vogel EM et al.; The metabolism of glucose and xylose as a function of oxygenation in Pichia stipitis and Saccharomyces cerevisiae cell suspensions was studied by 31P and 13C nuclear magnetic resonance spectroscopy . The rate of both glucose and xylose metabolism was slightly higher and the production of ethanol was slightly lower in aerobic than in anoxic cell suspensions of P . stipitis . As well, the cytoplasmic pH of oxygenated cells was more alkaline than that of nonoxygenated cells . In contrast, in S . cerevisiae, the intracellular pH and the rate of glucose metabolism and ethanol production were the same under aerobic and anoxic conditions . Agarose-immobilized Pichia stipitis was able to metabolize xylose or glucose for 24 to 60 h at rates and with theoretical yields of ethanol similar to those obtained with anoxic cell suspensions . Cell growth within the beads, however, was severely compromised . The intracellular pH {pH(int)} of the entrapped cells fell to more acidic pH values in the course of the perfusions relative to corresponding cell suspensions . Of importance was the observation that no enhancement in the rate of carbohydrate metabolism occurred in response to changes in the pH(int) value . In contrast to P . stipitis, agarose-immobilized Saccharomyces cerevisiae showed a dramatic twofold increase in its ability to metabolize glucose in the immobilized state relative to cell suspensions . This strain was also able to grow within the beads, although the doubling time for the entrapped cells was longer, by a factor of 2, than the value obtained for log-phase batch cultures . Initially, the pH(int) of the immobilized cells was more alkaline than was observed with the corresponding S . cerevisiae cell suspensions; however, over time, the intracellular pH became increasingly acidic . As with immobilized P . stipitis, however, the pH(int) did not play a key role in controlling the rate of glucose metabolism. Proteins, 1996 Jul, 25(3), 398 - 400 Crystallization and preliminary X-ray diffraction studies of human procathepsin L; Coulombe R et al.; Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na,K)PO4 buffer, pH 7.8 . Crystal size was increased by multiple macroseeding . The crystals are orthorhombic, of space group P2 1 2 1 2 1, with cell dimensions of a = 40.2 A, b = 88.4 A, and c = 94.9 A . A 2.2 A native data set was collected using synchrotron radiation . Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion . Heavy-atom derivative search is in progress. Yeast, 1996 Jul, 12(9), 815 - 22 Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris; Rodriguez L et al.; A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha . We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H . polymorpha yeast . The culture conditions for invertase production using a fed-batch culture were studied . More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space . The fermentative process was scaled up to 50 l . Invertase produced from H . polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S . cerevisiae . Using the Western-blot technique, it was observed that invertase secreted from H . polymorpha and invertase secreted from S . cerevisiae showed common antigenic determinants. EMBO J, 1996 Jul 1, 15(13), 3275 - 85 Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins; Kalish JE et al.; Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP) . These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris . Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes . Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins . While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein . These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane . Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane . In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity. Biochemistry, 1996 Jun 25, 35(25), 8149 - 57 Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases; Carmona E et al.; The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion) . Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L . The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC . The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide) . The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2 . The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide . The inhibitory activity was also found to be strongly pH-dependent . Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L . The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity. EMBO J, 1996 Jun 17, 15(12), 2914 - 23 The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor; Yahraus T et al.; In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases . We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris . Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD . Consistent with this observation, CG4 patients carry mutations in PXAAA1 . The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein . Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase . Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p . We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity. Biochem Mol Biol Int, 1996 Jun, 39(3), 471 - 85 Cloning, expression and characterization of a blood group B active recombinant alpha-D-galactosidase from soybean (Glycine max); Davis MO et al.; A cDNA encoding soybean alpha-D-galactosidase {E.C . 3.2.1.22} was obtained by screening a soybean library with Phaseolus alpha-D-galactosidase cDNA . The Glycine max alpha-D-galactosidase cDNA is 1.75 kb long and contains untranslated 5' and 3' sequences . The deduced amino acid sequence of the soybean gene has a high degree of homology with other eucaryotic alpha-D-galactosidases . Recombinant alpha-D-galactosidase (rGal) was expressed in Pichia pastoris and purified by affinity chromatography . Purified rGal was homogeneous as judged by SDS-PAGE analysis with the relative molecular mass under reducing conditions of 39.8, and under nonreducing conditions 38.0 kDa . The expressed protein contained the sequence NGLGHTPPMG at the N-terminus, corresponding to the deduced amino acid sequence of the soybean gene . The relative native molecular mass by Sephacryl S-200 chromatography was determined to be 33.1 kDa . The specific activity was 295.6 mumoles of PNP-alpha-D-galactopyranoside hydrolyzed per mg pure rGal per min . rGal was highly specific for alpha-D-galactosyl residues . No detectable hemagglutinin or protease activity was present in the preparations . Furthermore, rGal was active against the blood group B antigen in native human erythrocyte cell suspension assays . The only detectable erythrocyte phenotypic change was loss of the B and P1 epitopes . Consequently, recombinant Glycine max alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of universally transfusable type O erythrocytes by enzymatic conversion. Protein Expr Purif, 1996 Jun, 7(4), 423 - 30 Production and purification of human fibroblast collagenase (MMP-1) expressed in the methylotrophic yeast Pichia pastoris; Rosenfeld SA et al.; The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeast Pichia pastoris . The proMMP-1 encoding DNA was fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal in the P . pastoris pPIC9 expression plasmid, transformed into strain GS115 (His-), and His+ Muts (slow methanol utilization) transformants were selected . Full-length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter fermentations . The protein was purified to greater than 95% homogeneity . The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity . These data suggest that the P . pastoris expression system offers a convenient and efficient means to produce and purify MMP-1. J Histochem Cytochem, 1996 Jun, 44(6), 581 - 9 Labeling of peroxisomes with green fluorescent protein in living P . pastoris cells; Monosov EZ et al.; We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides . GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris . GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients . Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light . The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives . This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P . pastoris is cytosolic, whereas GFP-SKL is peroxisomal . The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis. Curr Genet, 1996 Jun, 30(1), 3 - 11 Moving pictures and pulsed-field gel electrophoresis show only linear mitochondrial DNA molecules from yeasts with linear-mapping and circular-mapping mitochondrial genomes; Jacobs MA et al.; The mobility of mitochondrial DNA (mtDNA) in pulsed-field gel electrophoresis (PFGE) and its appearance in moving pictures from fluorescence microscopy were used to investigate the mitochondrial genome structure for five Pichia and Williopsis strains of yeast . An apocytochrome b-gene hybridization probe identified only linear mtDNA molecules for each strain when total cellular DNA was fractionated by PFGE . Most of the mass of DNA isolated from mitochondria for one linear-mapping and one circular-mapping mitochondrial genome was found in linear molecules much larger than the genome size of 50 kb; some molecules were as long as 1500 kb, but only a trace amount of apparently circular mtDNA was found for the strain with the circular-mapping genome . Probes for both the apocytochrome-b and mitochondrial small rRNA subunit genes hybridized strongly to mtDNA of approximately 50-100 kb, but weakly to the larger DNA from mitochondria of these two strains . For the four linear-mapping strains, PFGE revealed two or three distinct bands of linear mtDNA, larger than the genome size, within a smear of approximately 50-100 kb, but a smear without bands was found for the circular-mapping strain. Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5357 - 62 Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins; Lima CD et al.; The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering . The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta . The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides . PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans . Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo . The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices . Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer . PKCI-1 has been shown to interact specifically with zinc . The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms . The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature. Ann N Y Acad Sci, 1996 May 15, 782, 555 - 65 Molecular cloning of a lytic beta-1,3-glucanase gene from Oerskovia xanthineolytica LLG109 . A beta-1,3-glucanase able to selectively permeabilize the yeast cell wall; Ferrer P et al.; Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B . subtilis . This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S . cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast . Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O . xanthineolytica LLG109 . The amplified fragment has been cloned and sequenced . The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O . xanthineolytica LLG109 . By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O . xanthineolytica LLG109 . All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions . The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids . The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced . The cloning of the lytic beta-1,3-glucanase from O . xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned . The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation . In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S . cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology. Yeast, 1996 May, 12(6), 541 - 53 Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product; Tsujikawa M et al.; Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR) . The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0.47 mg/l; however, most of the secreted product had been processed to smaller polypeptides . The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA . Some approaches to minimizing the proteolysis of scu-PA-Q302 were attempted . Addition of Triton X-100, L-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q302 and increased the yield of immunoreactive material to approximately 5 mg/l . Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation . Co-expression of scu-PA-Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis . Scu-PA-Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose . The purified protein had a molecular weight of 47 kDa . It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain . N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast . Scu-PA-Q302 closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties. Biosci Biotechnol Biochem, 1996 May, 60(5), 818 - 22 Should Petasospora BOIDIN et ABADIE (Saccharomycetaceae) be retained?--the phylogeny based on the partial sequences of 18S and 26S ribosomal RNAs; Yamada Y et al.; The six strains of the Pichia species, once classified in the genus Petasospora, were examined for their 18S (positions 1451-1618, 168 bases) and 26S (positions 1611-1835, 225 bases and 493-622, 130 bases) rRNA partial base sequencings . In the 18S rRNA partial base sequencings, the type species of the genus Petasospora (Petasospora rhodanensis) was found to be closely related to Pichia anomala (identical to Hansenula anomala, type species of genus Hansenula) with base differences of two, but not to Pichia membranaefaciens, the type species of the genus Pichia, with base differences of ten . However, the species was not so closely related to P . anomala in the 26S rRNA partial base sequencings with base differences of twelve and 68 percent similarity . The genus Petasospora was known to be a very heterogeneous taxon phylogenetically with base differences of thirty-one to three and ninety-two to eight and with 40 to 83 percent similarities . The sequence data obtained here and the phenotypic features described previously indicate that the type species of the genus Petasospora (Pet . rhodanensis, identical to Pichia rhodanensis) is adequate to be accommodated temporarily in the genus Pichia, a heterogeneous taxon, until the precise taxonomic position of the type species is defined. Appl Environ Microbiol, 1996 May, 62(5), 1839 - 41 Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli; Graupner S et al.; Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid . Evidence that plasmid-like structures are formed in H . polymorpha and that their formation is stimulated by DNA damage is presented in this study. Mol Cell Biol, 1996 May, 16(5), 2527 - 36 The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1; Waterham HR et al.; We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris . The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif . Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina . In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1 . Like PAF-1, Per6p is a peroxisomal integral membrane protein . In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent . Instead, peroxisomal remnants are observed . In addition, peroxisomal matrix proteins are synthesized but located in the cytosol . The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1. Int J Food Microbiol, 1996 Apr, 29(2-3), 167 - 75 Media for preservative resistant yeasts: a collaborative study; Hocking AD; An international collaborative study was carried out to determine the most effective medium for selective isolation and enumeration of preservative resistant yeasts . Such a medium should prevent the growth of other yeasts such as Saccharomyces cerevisiae that are tolerant to lower levels of commonly used food preservatives, and sensitive yeasts such as Rhodotorula species . The study compared two non-selective media that are in common use for cultivation of yeasts from foods, Malt Extract agar (MEA) and Tryptone Glucose Yeast extract agar (TGY) with media made selective for preservative resistant yeasts by addition of 0.5% acetic acid to these two basal media (MEAA and TGYA) . A fifth medium, Zygosaccharomyces bailii medium (ZBM) was also included in the study . These media were compared for their efficacy in selective isolation and enumeration of the preservative resistant yeasts Zygosaccharomyces bailii, Schizosaccharomyces pombe and Pichia membranaefaciens . MEA and TGY without acetic acid were used as control, non-selective media, and Rhodotorula glutinis was the preservative sensitive control culture . Seven laboratories in six countries took part in the study . Of the non-selective media, TGY generally gave the highest counts, and TGY amended with 0.5% acetic acid (TGYA) was the best medium for recovery of all three preservative-resistant yeasts . ZBM was found to be selective for Z . bailii, but counts of this yeast on ZBM were significantly lower than on TGYA . R . glutinis did not grow on any of the selective media. Biochem Biophys Res Commun, 1996 Mar 18, 220(2), 372 - 6 Inhibition of Aspergillus serine proteinase by Streptomyces subtilisin inhibitor and high-level expression of this inhibitor in Pichia pastoris; Markaryan A et al.; Aspergillus fumigatus encodes an extracellular serine proteinase of the subtilisin family that is thought to be involved in invasive aspergillus infection of immunocompromised patients . When the structure of proteinase K was used to model this Aspergillus serine proteinase, Streptomyces subtilisin inhibitor (SSI) was predicted to be capable of binding to the serine proteinase . SSI purified from S . albogriseolus inhibited the serine proteinase with Ki of 1 x 10(-9)M . This value is higher than that for subtilisin probably because the serine proteinase lacks the S(4-6) site that interacts with the P(4-6) site of SSI . A high level expression for SSI, established in Pichia pastoris, yielded 0.5g of SSI per liter of culture medium . The secreted product was easily purified to homogeneity and biochemically characterized . The recombinant SSI showed a Ki of 1.1 x 10(-9) and 1 x 10(-8) for the serine proteinases from A . fumigatus and A . flavus, respectively. Eur J Biochem, 1996 Mar 15, 236(3), 978 - 83 Influence of expression system on chromophore binding and preservation of spectral properties in recombinant phytochrome A; Gartner W et al.; N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation . The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39-kDa and 59-kDa peptides, respectively . The absorption properties and photochromicity upon red/far-red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N-termini, but C-terminal positions identical to those of the corresponding tryptic fragments (45-kDa and 65-kDa peptides) . All recombinant 65-kDa and 59kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the Pr and the Pfr forms . The smaller chromopeptides (45-kDa and 39-kDa) behaved differently depending on the expression system employed . E . coli-derived peptides exhibited a phytochrome-like difference spectrum only when the intact N-terminus was present (45-kDa product) . The recombinant 39-kDa peptide from E . coli was incapable of chromophore binding whereas the identical peptide sequence expressed by P . pastoris formed a chromoprotein with phycocyanobilin . This recombinant phytochrome fragment exhibited a difference spectrum (Pr-Pfr) with an even larger Pfr absorption band than the comparable tryptic 39-kDa fragment . Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics . Evidently, trypsin digestion down to the 39-kDa fragment affects protein conformation also in terms of Pfr conservation. J Biol Chem, 1996 Mar 15, 271(11), 6490 - 6 Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis; Brandes HK et al.; Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner . Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host . To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene . The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme . This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart . Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250 . Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate . Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme . Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation. J Biol Chem, 1996 Mar 15, 271(11), 6298 - 305 Developmental expression of a tandemly repeated, proline-and glutamine-rich amino acid motif on hyphal surfaces on Candida albicans; Staab JF et al.; cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS . The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues . The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris . The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues . The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms . These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces . The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins. Arch Biochem Biophys, 1996 Mar 15, 327(2), 324 - 9 Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes; Zhu A et al.; Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells . Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials . Recently we expressed the recombinant alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column . Purified (r)alpha-GAL, migrating as a single band of 41 kDa on a SDS-PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (K(m) =0.363 mM and V(max) = 46.9 U/mg) . Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substrate p-nitrophenol-alpha-D-galactopyranoside . Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface . In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies . Thus, with a simple procedure for over-expression and purification of (r)alpha-GAL from P . pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells. J Lipid Res, 1996 Mar, 37(3), 599 - 605 Expression and secretion of rabbit plasma cholesteryl ester transfer protein by Pichia pastoris; Kotake H et al.; The rabbit cholesteryl ester transfer protein (CETP) was expressed in the methylotrophic yeast Pichia pastoris by introducing the CETP cDNA under the control of the methanol-inducible alcohol oxidase promoter . The cDNA was cloned from in vitro amplified cDNA of rabbit liver mRNA . The nucleotide sequence of the cloned cDNA differed slightly from the previously published sequence that changed the amino acid sequence in six residues . Interestingly, five of these replacements are identical to the corresponding residues in human CEPT . In addition, the encoded mature N-terminal sequence was changed from Cys- to Arg-Glu-Phe- to link the CETP sequence to the yeast acid phosphatase signal peptide . The culture medium of the transformed cells induced with 1% methanol contained both cholesteryl ester and triglyceride transfer activity comparable to that of rabbit plasma . Like rabbit plasma, the lipid transfer activity in the medium could be inhibited by monoclonal antibodies that block CE/TG transfer or TG transfer alone . Immunoblot analysis of M(r) = 80 K and minor species of M(r) = 60-100 K . In spite of these differences, the specific transfer activity of the recombinant CETP was indistinguishable from that of rabbit plasma CETP of M(r) = 74 K . N-Glycosidase F treatment converted both the recombinant and plasma CETP to a single species of M(r) = 55 K . Both the plasma and recombinant CETP lost their activity after removal of N-linked carbohydrate and sialic acid . A single 55 K component was found in the cell-lysates . The intracellular form of the recombinant CETP was not modified by N-glycosidase F treatment . In conclusion, the recombinant CETP is synthesized as an inactive polypeptide that is processed and secreted as a functional glycoprotein . In addition, the N-terminal Cys residue of the plasma CETP is not required for its activity. Plant Cell, 1996 Mar, 8(3), 519 - 27 Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase; Su W et al.; Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels . To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed . Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis . Active NR protein was produced in a Pichia pastoris expression system . Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA . Mutant forms of NR were constructed, produced in P . pastoris, and tested in the in vitro inactivation assay . Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach . A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect . The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated . These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro. J Immunol, 1996 Mar 1, 156(5), 1880 - 5 Human natural yeast killer toxin-like candidacidal antibodies; Polonelli L et al.; A murine mAb (mAbKT4, IgG1) that neutralized in vitro the anti-Candida activity of a killer toxic (KT) from the yeast Pichia anomala acted as an idiotypic (Id) vaccine in eliciting anti-Id Abs with toxin-like activity (KT-IdAb) in a rat vaginitis model . In this study, we demonstrate that intravaginal or intragastric inoculations of Candida albicans bearing a receptor for the toxin was able to recall KT-IdAb production in the vagina of the animals primarily immunized with mAbKT4 and also to elicit by themselves an Ab that functionally mimicked the KT (KTAb) . Anti-Id-like, KT-like Abs were also consistently found in the vaginal fluid of human vaginitis patients who were infected by Candida but who had never been exposed to the Id vaccine . These Abs were as candidacidal in vitro as those raised in rat vagina by the Id vaccination, and, likewise, their cytocidal effect was totally neutralized by previous reaction with mAbKT4 . Importantly, they were also able to confer a significant anticandidal protection in the rat vaginitis model, comparable to that achievable by KT-IdAb passively transferred to naive rats from Id-vaccinated animals . Thus, candidacidal Abs representing the internal image of a yeast KT are part of the Ab repertoire that follows infection or immunization with Candida . It is speculated that the host's immune system response may exploit the KT receptor of microbial pathogens to produce microbicidal Abs, possibly mirroring competition events among microorganisms in natural habitats. Biotechnol Appl Biochem, 1996 Feb, 23 ( Pt 1), 23 - 8 Biochemical characterization of the recombinant Boophilus microplus Bm86 antigen expressed by transformed Pichia pastoris cells; Montesino R et al.; In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris . The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm . The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein. J Interferon Cytokine Res, 1996 Feb, 16(2), 119 - 26 High yield expression and secretion of the ovine pregnancy recognition hormone interferon-tau by Pichia pastoris; Van Heeke G et al.; The early conceptus (embryo and associated membranes) of domestic ruminats signals its presence to the maternal uterus through production of interferon-tau (IFN-tau) . Production of IFN-tau ensures continued production of progesterone, the hormone of pregnancy, by the ovarian corpus luteum . This paper reports the high-level expression and efficient secretion of biologically active recombinant ovine IFN-tau (rOvIFN-tau) by Pichia pastoris . The developed method produces more than 80% pure recombinant ovine IFN-tau, obviating the need for further purification for many purposes . Initial fermentation studies produced IFN-tau at 280 mg/liter and demonstrate the potential of this system for large-scale production of IFN-tau. Toxicon, 1996 Feb, 34(2), 213 - 24 Facile production of native-like kappa-bungarotoxin in yeast: an enhanced system for the production of a neuronal nicotinic acetylcholine receptor probe; Fiordalisi JJ et al.; Research on the mammalian central nervous system had been hindered by the limited number and meager supply of naturally occurring toxins that can be used as pharmacological reagents . The kappa-neurotoxins in particular are not found abundantly in nature and are difficult to obtain and isolate in quantities sufficient for research purposes . Here we report the expression and isolation of relatively large quantities of the kappa-neurotoxin, kappa-bungarotoxin, in an active form using a yeast, Pichia pastoris, expression system . The resultant product of the expression system has a short amino-terminal amino acid extension relative to venom-derived kappa-bungarotoxin, but is equivalent to the native toxin in physical and biological properties, as judged by the CD spectra, the ability to form dimers in solution, and the activity on chick ciliary ganglia . The yeast system produces approximately 0.2 mg from a 2 liter culture and the purification takes approximately 2 days . In contrast, E . coli, the only other available expression system for this toxin, produces one-fifth to one-half as much active material from a 5 liter high-density fermentation and the resulting protein takes over a week to purify . No high mol . wt disulfide-bonded aggregates were found in the yeast expression system product, indicating that the product is that of a biologically assisted folding process . This has significant implications not only for the efficient production of native toxin but also for the production of mutant proteins to study the structure-function relationship in these proteins. Arch Biochem Biophys, 1996 Feb 1, 326(1), 8 - 14 Functional expression of recombinant spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase/C17,20-lyase) in yeast (Pichia pastoris); Trant JM; The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 (CYP17) was used to direct the heterologous expression of a functional enzyme in yeast (Pichia pastoris) . This protein possesses two enzymatic activities: 17 alpha-hydroxylase and C17,20-lyase reactions . Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue . This study describes the culture conditions and the enzymatic activity of recombinant shark cytochrome P450c17 . The shark enzyme was compatible with the endogenous yeast NADPH-cytochrome P450 reductase and was bioactive within the living yeast cell . Progesterone (at 15 microM) was metabolized (51 pmol/min/10(9) cells) faster than pregnenolone (36 pmol/min/10(9) cells) . Both progesterone and pregnenolone were completely metabolized to their respective androgens (androstenedione and dehydroepiandrosterone) . Although 11 beta-hydroxy-progesterone was readily 17 alpha-hydroxylated by the shark P450, the lyase reaction was not evident . Alterations to the 2-carbon sidechain of progesterone (21-hydroxylation or 20 beta-reduction) prevented metabolism . High-density cultures (> 1.5 x 10(9) cells/ml) yielded the greatest quantity of recombinant protein but cultures of lower density produced more recombinant protein per cell . This is the first report of heterologous expression in yeast of a steroidogenic cytochrome P450 from a lower vertebrate. Adv Exp Med Biol, 1996, 409, 147 - 55 Recombinant expression and epitope mapping of grass pollen allergens; Suphioglu C et al.; We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens . Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients . Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus . However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE . We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding . cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding . Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes . The allergenic epitopes were localised towards the C-terminal half of the molecule . Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7 . At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens . These results aid in the design of diagnostics and in grass pollen immunotherapy. Mycopathologia, 1996, 135(1), 1 - 8 Killer factor interference in mixed opportunistic yeast cultures; Conti S et al.; The interaction of the killer yeast Pichia anomala UP 25F with the killer toxin-sensitive clinical isolate Candida albicans UCSC 10S and its natural toxin-resistant mutant derivative C . albicans UCSC 10R were studied under various conditions . A differential inhibition was shown to occur in vitro at pH and temperature values, which are not encountered in vivo, only by using preformed killer toxin, since antagonism due to yeast growth proved to be predominant on the killer effect . Under adverse growth conditions, the P . anomala killer yeast proved to be able to produce an anatoxin antigenically related to the active or heat inactivated killer toxin . These findings suggest that killer toxins may not function as potential virulence factors in the competition between the opportunistic killer yeast P . anomala and sensitive microorganisms for colonization in the course of natural human infections. Parasitol Res, 1996, 82(2), 114 - 6 Inhibitory effect of a Pichia anomala killer toxin on Pneumocystis carinii infectivity to the SCID mouse; Seguy N et al.; A Pichia anomala killer toxin has been demonstrated to have a specific inhibitory effect on the in vitro attachment of Pneumocystis carinii . The results presented herein show that this yeast toxin is also effective against P . carinii infectivity in reducing parasite colonization in the lungs of SCID mice . The specificity of this inhibitory effect was controlled using a monoclonal antibody neutralizing the killer properties of the yeast toxin. Yeast, 1996 Jan, 12(1), 31 - 40 Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y; Ohi H et al.; We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene . Using the S . cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P . pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned . The open reading frame of the P . pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids . The molecular mass of the protein is calculated to be 59.44 kDa without sugar chains . The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites . The NH2-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant . There is 61% identity between the amino acid sequences of P . pastoris Prc1p and S . cerevisiae Prc1p . Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity . Over-expression of the PRC1 gene under regulation of the P . pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY . The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987. Appl Biochem Biotechnol, 1996 Spring, 57-58, 267 - 76 Increased xylose reductase activity in the xylose-fermenting yeast Pichia stipitis by overexpression of XYL1; Dahn KM et al.; The Pichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid that P . stipitis maintains in multicopy . The plasmid pXOR with the XYL1 insert or a control plasmid pJM6 without XYL1 was introduced into P . stipitis . When grown on xylose under aerobic conditions, the strain with pXOR had up to 1.8-fold higher xylose reductase (XOR) activity than the control strain . Oxygen limitation led to higher XOR activity in both experimental and control strains grown on xylose . However, the XOR activities of the two strains grown on xylose were similar under oxygen limitation . When grown on glucose under aerobic or oxygen-limited conditions, the experimental strain had XOR activity up to 10 times higher than that of the control strain . Ethanol production was not improved, but rather it decreased with the introduction of pXOR compared to the control, and this was attributed to nonspecific effects of the plasmid. Appl Biochem Biotechnol, 1996 Spring, 57-58, 201 - 12 Peculiarities of the regulation of fermentation and respiration in the crabtree-negative, xylose-fermenting yeast Pichia stipitis; Passoth V et al.; The respiration of Pichia stipitis was not repressed by either high concentrations of fermentable sugars or oxygen limitation . Fermentation was not induced by high sugar concentrations, but was inactivated by aerobic conditions . The activity of pyruvate dehydrogenase was constitutive . In contrast, pyruvate decarboxylase, alcohol dehydrogenase, and aldehyde dehydrogenase were induced by a reduction in the oxygen tension . It was demonstrated that in P . stipitis, the pyruvate decarboxylase is not induced by a signal from glycolysis . Contrary to Saccharomyces cerevisiae, the pyruvate decarboxylase was not inhibited by phosphate. Microbiology, 1996 Jan, 142 ( Pt 1), 165 - 72 A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae; Meinander N et al.; Recombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates . At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed . The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism . At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased . The specific ethanol flux was not affected . At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased . The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction . Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol . Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production . The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes . Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate . At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased . Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products . Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted . However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance. Gene, 1995 Dec 29, 167(1-2), 215 - 9 High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: engineering a biocatalyst; Payne MS et al.; Glycolate oxidase (GO) is a flavo-enzyme that catalyzes the oxidation of glycolate, and is useful for the biocatalytic production of glyoxylate . We have produced high levels of spinach GO in the methylotrophic yeast Pichia pastoris (Pp), by chromosomal integration of multiple copies of an expression cassette containing the GO coding sequence under control of the methanol-inducible alcohol oxidase I promoter . Under fermentation conditions, greater than 250 units of GO per gram of cells (wet weight) was obtained, corresponding to roughly 20-30% of soluble cell protein . This recombinant Pp strain was used as a whole-cell biocatalyst for conversion of glycolic acid to glyoxylic acid. FEBS Lett, 1995 Dec 27, 377(3), 451 - 6 Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization; Weiss HM et al.; The methylotrophic yeast Pichia pastoris was tested for heterologous expression of the mouse 5-HT5A receptor . Three different expression plasmids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promoter of the P . pastoris alcohol oxidase 1 (AOX1) gen . The expression plasmids differed with respect to the signal sequences used for N-terminal fusion . In two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor . Expression of functional receptor after transformation of strain GS115 was detected by radioligand binding using {3H}LSD . The construct with the best expression levels in strain GS115 was used for transformation of the protease deficient strain SMD1163 . Here, the expression level was 2-8 times higher . Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of {3H}LSD with a Kd of approximately 1.9 nM . Receptor concentrations of approximately 22 pmol/mg membrane protein revealed the potential of the P . pastoris expression system for high level expression of membrane proteins . The pharmacological properties were comparable to those reported for the receptor expressed in mammalian systems. Protein Expr Purif, 1995 Dec, 6(6), 813 - 20 Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris; Brankamp RG et al.; Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii . In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments . A yeast expression plasmid, pPIC9HG-2, was constructed by making an inframe fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion . The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter . Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination . Both strains yielded His+ transformants that secreted a potent anticoagulant activity into the medium . Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone . The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71 . Significant clonal variation in the expression of r-ghilanten was found among the His+ transformants . A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales . r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography . Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 314 - 20 Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains; Yu S et al.; Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l . When xylulose was the sole carbon source and fermentation was anaerobic, S . cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells-1 h-1 compared with 0.833 gg cells-1 h-1 for glucose . Bakers' yeast and S . cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains . While P . stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C . shehatae NJ 23 fermented xylulose at a rate of 0.345 gg cells-1 h-1, compared with 0.575 gg cells-1 h-1 for glucose . For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations . When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S . cerevisiae ATCC 24860 and C . shehatae NJ 23, and 50%- 65% of the assimilated carbon could not be accounted for in the products determined. Appl Environ Microbiol, 1995 Dec, 61(12), 4251 - 7 Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp; Morais PB et al.; The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined . The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit . The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest . The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin . Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell . These yeasts were dispersed and served as food for the invader Drosophila malerkotliana . The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell . The killer toxin-producing yeasts Pichia kluyveri var . kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration . An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition . The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate. Appl Environ Microbiol, 1995 Dec, 61(12), 4184 - 90 Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase; Walfridsson M et al.; Saccharomyces cerevisiae was metabolically engineered for xylose utilization . The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S . cerevisiae . The gene products catalyze the two initial steps in xylose utilization which S . cerevisiae lacks . In order to increase the flux through the pentose phosphate pathway, the S . cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed . A XYL1- and XYL2-containing S . cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2 . Overexpression of only TKL1 did not influence growth . The results indicate that the transaldolase level in S . cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites . Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL . The rate of xylose consumption was higher in the presence of glucose . Xylose was used for growth and xylitol formation, but not for ethanol production . Decreased oxygenation resulted in impaired growth and increased xylitol formation . Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation. J Bacteriol, 1995 Dec, 177(24), 7070 - 7 Cloning of a novel constitutively expressed pectate lyase gene pelB from Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris; Guo W et al.; Since plant-pathogenic fungi must penetrate through pectinaceous layers of the host cell wall, pectin-degrading enzymes are thought to be important for pathogenesis . Antibodies prepared against a pectin-inducible pectate lyase (pectate lyase A {PLA}) produced by a phytopathogenic fungus, Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI), was previously found to protect the host from infection . The gene (pelA) and its cDNA were cloned and sequenced . Here we report the isolation of a new pectate lyase gene, pelB, from a genomic library of F . solani f . sp . pisi with the pelA cDNA as the probe . A 2.6-kb DNA fragment containing pelB and its flanking regions was sequenced . The coding region of pelB was amplified by reverse transcription-mediated PCR, using total RNA isolated from F . solani pisi culture grown in the presence of glucose as the sole carbon source . The predicted open reading frame of pelB would encode a 25.6-kDa protein of 244 amino acids which has 65% amino acid sequence identity with PLA from F . solani f . sp . pisi but no significant homology with other pectinolytic enzymes . The first 16 amino acid residues at the N terminus appeared to be a signal peptide . The pelB cDNA was expressed in Pichia pastoris, yielding a pectate lyase B (PLB) which was found to be a glycoprotein of 29 kDa . PLB was purified to homogeneity by using a two-step procedure involving ammonium sulfate precipitation followed by Superdex G75 gel filtration chromatography . Purified PLB showed optimal lyase activity at pH 10.0 . A rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLB cleaved polygalacturonate chains in an endo fashion . Western blotting (immunoblotting) with antibodies raised against PLA showed that PLB and PLA are immunologically related to each other . The 5' flanking regions of both pelA and pelB were translationally fused to the beta-glucuronidase gene and introduced into F . solani f . sp . pisi, and beta-glucuronidase activities of the transformants were measured . Expression of the marker gene by the transformants showed that pelA expression is induced by pectin and repressed by glucose, whereas expression of pelB is constitutive and is not subject to glucose repression . Reverse transcription-mediated PCR showed that both pelA and pelB are expressed when F . solani f . sp . pisi infects pea epicotyl. Arch Biochem Biophys, 1995 Dec 1, 324(1), 65 - 70 High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris; Zhu A et al.; alpha-Galactosidase isolated from coffee beans cleaves the terminal alpha-galactose residues from oligosaccharide chains on blood group B red cells, thus generating group O cells . Such enzymatically converted red cells not only maintain full erythrocyte integrity and viability in vitro, but also demonstrate immune tolerance and a normal life span in vivo . In order to produce large quantities of recombinant alpha-galactosidase for use in the study of blood-type conversion, we subcloned the cDNA coding for coffee bean alpha-galactosidase into the EcoRI site of the vector pPIC9 in order to express the enzyme in Pichia pastoris, a methylotrophic yeast strain . After P . pastoris transformation, colonies were screened for high-level expression of alpha-galactosidase, based on enzyme activity . In order to increase enzyme production, the growth conditions in the shake flask culture and fermentor culture were optimized . Under the conditions applied, biologically active alpha-galactosidase was produced and secreted into the culture medium at a level of approximately 0.4 g per liter of the fermentor culture . The protein was purified to apparent homogeneity by a simple chromatography procedure, as suggested by a single band of 41 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Its homogeneity was further confirmed by chromatofocusing and N-terminal sequencing . P . pastoris appears to be the choice as host for the large-scale production of recombinant alpha-galactosidase used for blood type conversion. Arch Biochem Biophys, 1995 Nov 10, 323(2), 352 - 60 Cloning of a new pectate lyase gene pelC from Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) and characterization of the gene product expressed in Pichia pastoris; Guo W et al.; Antibodies prepared against a pectate lyase (PLA) produced by a phytopathogenic fungus Fusarium solani f . sp . pisi (Nectria haematococca, mating type VI) were previously found to protect the host against infection . The cDNA and gene (pelA) for PLA were cloned and sequenced . A new pectate lyase gene, pelC, was isolated from a genomic library of F . solani pisi with pelA cDNA as a probe . A 1.3-kb DNA fragment containing the pelC gene and its flanking regions was identified and sequenced . The coding region of pelC was amplified by reverse transcription-polymerase chain reaction using total RNA isolated from a pectin-induced F . solani pisi culture as template . The open reading frame of pelC was predicted to encode a 23.3-kDa protein of 219 amino acid residues, which shares 51% identity with PLA from F . solani pisi . No typical fungal leader peptide sequence could be identified at the N-terminus of the predicted protein sequence . The amplified pelC cDNA was expressed in Pichia pastoris yielding a pectate lyase C (PLC) with a molecular mass of 26.0 kDa and containing carbohydrates . PLC was purified to homogeneity using Mono Q anion-exchange chromatography . Purified PLC required Ca2+ for its activity and showed optimal lyase activity at pH 9.5 and 55 degrees C . Rapid drop in the viscosity of the substrate and Mono Q anion-exchange chromatography of the products generated by the lyase showed that PLC cleaved polygalacturonate chains in an endo fashion . Western blot using antibodies raised against PLA and PLC showed that PLC and PLA are immunologically related to each other. Protein Eng, 1995 Nov, 8(11), 1177 - 87 Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388; White CE et al.; Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity . Fermentation of P . pastoris resulted in yields of 170 mg/l TM . Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained . Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains . These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM . The fragment containing the fourth and fifth EGF-like domains {TMEGF(4-5)} had 10% the specific activity of rabbit TM . Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain . Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments. Anal Biochem, 1995 Nov 1, 231(2), 342 - 8 Concanavalin A- and wheat germ agglutinin-conjugated lectins as a tool for the identification of multiple N-glycosylation sites in heterologous protein expressed in yeast; Garcia R et al.; We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase . Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein . Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry . This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris. J Interferon Cytokine Res, 1995 Nov, 15(11), 955 - 63 Expression of a human mutant monocyte chemotactic protein 3 in Pichia pastoris and characterization as an MCP-3 receptor antagonist; Masure S et al.; The cDNA encoding human monocyte chemotactic protein 3 (hMCP-3) was cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris . After transformation of P . pastoris by electroporation, several clones with