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Pastoris
Kinds of Pastoris Terms modified by Pastoris Selected AbstractsCopper is required for prion protein-associated superoxide dismutase-l activity in Pichia pastorisFEBS JOURNAL, Issue 5 2007Carina Treiber The prion protein (PrP) is the key protein implicated in transmissible spongiform encephalopathies. It is a metalloprotein that binds manganese and copper. The latter is involved in the physiological function of the protein. We have previously found that PrP expression in Pichia pastoris affects intracellular metal ion concentrations and that formation of protease-resistant PrP is induced by additional copper and/or manganese. In this study, we show that heterologously expressed PrP is post-translationally modified and transported to the cell wall. We found by combining three different test systems that PrP itself had gained superoxide dismutase-like activity in P. pastoris. However, this activity could not be inhibited by KCN and depended on additional copper in the medium. Thus, this study defines the conditions under which PrP exhibits superoxide dismutase-like activity by showing that copper must be present for the protein to participate in scavenging and detoxification of reactive oxygen species. [source] Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastorisFEBS JOURNAL, Issue 6 2000Ludovic Otterbein Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae,-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated. [source] Assessment of amino-acid substitutions at tryptophan 16 in ,-galactosidaseFEBS JOURNAL, Issue 5 2000Elizabeth Maranville The tryptophan residue at position 16 of coffee bean ,-galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site-directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known. [source] Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplementFEMS MICROBIOLOGY LETTERS, Issue 1 2010Piyanun Harnpicharnchai Abstract Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50,55 °C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed. [source] Functional importance of Asp37 from a family 11 xylanase in the binding to two proteinaceous xylanase inhibitors from wheatFEMS MICROBIOLOGY LETTERS, Issue 1 2004Tariq A. Tahir Abstract Aspergillus niger xylanase is a target enzyme of the two wheat proteinaceous inhibitors, XIP-I and TAXI-I. We previously suggested that the xylanase "thumb" region was XIP-I binding site. Here, we expressed the Asp37Ala mutant in Pichia pastoris and showed that the mutation abolished the enzyme capacity to interact with both inhibitors, suggesting a direct contact at the active site. The mutant pH profile was altered, confirming the key role of Asp37 in determining the pH optima of glycoside hydrolase family 11. The results are consistent with a competitive inhibition mode and underline the strategic importance of Asp37 in the inhibition mechanism. [source] Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastorisFEMS YEAST RESEARCH, Issue 6 2009Tamara Wriessnegger Abstract Genetic manipulation of lipid biosynthetic enzymes allows modification of cellular membranes. We made use of this strategy and constructed mutants in phospholipid metabolism of Pichia pastoris, which is widely used in biotechnology for expression of heterologous proteins. Here we describe identification of two P. pastoris phosphatidylserine decarboxylases (PSDs) encoded by genes homologous to PSD1 and PSD2 from Saccharomyces cerevisiae. Using P. pastoris psd1, and psd2, mutants we investigated the contribution of the respective gene products to phosphatidylethanolamine synthesis, membrane composition and cell growth. Deletion of PSD1 caused loss of PSD activity in mitochondria, a severe growth defect on minimal media and depletion of cellular and mitochondrial phosphatidylethanolamine levels. This defect could not be compensated by Psd2p, but by supplementation with ethanolamine, which is the substrate for the cytidine diphosphate (CDP),ethanolamine pathway, the third route of phosphatidylethanolamine synthesis in yeast. Fatty acid analysis showed selectivity of both Psd1p and Psd2p in vivo for the synthesis of unsaturated phosphatidylethanolamine species. Phosphatidylethanolamine species containing palmitic acid (16:0), however, were preferentially assembled into mitochondria. In summary, this study provides first insight into membrane manipulation of P. pastoris, which may serve as a useful method to modify cell biological properties of this microorganism for biotechnological purposes. [source] Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomenaFEMS YEAST RESEARCH, Issue 2 2004Roland Weis Abstract Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate. [source] Momordica charantia trypsin inhibitor II inhibits growth and development of Helicoverpa armigeraINSECT SCIENCE, Issue 5 2009Manasi Alok Telang Abstract, Bitter gourd (Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs), which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera. In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-II, its cloning and expression as a recombinant protein using Pichia pastoris have been reported. Recombinant McTI-II inhibited bovine trypsin at 1: 1 molar ratio, as expected, but did not inhibit chymotrypsin or elastase. McTI-II also strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H. armigera larvae. The insect larvae fed with McTI-II-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding. Moreover, ingestion of McTI-II resulted in 23% mortality in the larval population. The strong antimetabolic activity of McTI-II toward H. armigera indicates its probable use in developing insect tolerance in susceptible plants. [source] An evaluation of garlic lectin as an alternative carrier domain for insecticidal fusion proteinsINSECT SCIENCE, Issue 6 2008Elaine Fitches Abstract The mannose-binding lectin GNA (snowdrop lectin) is used as a "carrier" domain in insecticidal fusion proteins which cross the insect gut after oral ingestion. A similar lectin from garlic bulb, ASAII, has been evaluated as an alternative "carrier". Recombinant ASAII delivered orally to larvae of cabbage moth (Mamestra brassica; Lepidoptera) was subsequently detected in haemolymph, demonstrating transport. Fusion proteins comprising an insect neurotoxin, ButaIT (Buthus tamulus insecticidal toxin; red scorpion toxin) linked to the C-terminal region of ASAII or GNA were produced as recombinant proteins (GNA/ButaIT and ASA/ButaIT) by expression in Pichia pastoris. In both cases the C-terminal sequence of the lectin was truncated to avoid post-translational proteolysis. The GNA-containing fusion protein was toxic by injection to cabbage moth larvae (LD50, 250 ,g/g), and when fed had a negative effect on survival and growth. It also decreased the survival of cereal aphids (Sitobion avenae; Homoptera) from neonate to adult by >70% when fed. In contrast, the ASA-ButaIT fusion protein was non-toxic to aphids, and had no effect on lepidopteran larvae, either when injected or when fed. However, intact ASA-ButaIT fusion protein was present in the haemolymph of cabbage moth larvae following ingestion, showing that transport of the fusion had occurred. The stabilities of GNA/ButaIT and ASA/ButaIT to proteolysis in vivo after injection or ingestion differed, and this may be a factor in determining insecticidal activities. [source] Cell growth and Trametes versicolor laccase production in transformed Pichia pastoris cultured by solid-state or submerged fermentationsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 4 2010Marcos López Abstract BACKGROUND: Growth kinetics of Pichia pastoris and heterologous expression of Trametes versicolor laccase were compared. This is the first study of its kind between solid-state yeast cultures done on polyurethane foam (PUF) and submerged liquid fermentations (SmF). RESULTS: The maximum values of biomass were similar for SSF (solid-state fermentation) and SmF experiments when the BOD (biochemical oxygen demand) was lower than 100 g L,1. For higher BOD levels, the maximum values of biomass were 55 g L,1 (SSF) and 35 g L,1 (SmF). Micrographs of PUF preparations showed yeast growing within liquid lamellae, thinner than 100 µm, forming large horizontal aggregates. Yeast aggregates were much smaller in SmF than in SSF experiments; however, laccase expression was lower in PUF than in SmF, unless the methanol concentration was increased to 63 g L,1, which was inhibitory only to the SmF system. CONCLUSION: The results show that oxygen mass transfer is more efficient in SSF, which is related to the higher area/volume ratio compared with SmF. Induction differences may also be due to hindered diffusion of methanol within large yeast aggregates. Copyright © 2009 Society of Chemical Industry [source] Comparative enzymology of native and recombinant house dust mite allergen Der p 1ALLERGY, Issue 3 2009J. Zhang Background:, The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. Methods:, Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. Results:, Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. Conclusion:, The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens. [source] Bayesian inference of evolutionary history from chloroplast microsatellites in the cosmopolitan weed Capsella bursa - pastoris (Brassicaceae)MOLECULAR ECOLOGY, Issue 14 2005ALF CEPLITIS Abstract Besides showing an extraordinary degree of phenotypic variability, Capsella bursa-pastoris (Brassicaceae) is also one of the world's most common plant species and a serious weed in many countries. We have employed a coalescent-based Bayesian analysis of chloroplast microsatellite data to infer demographic and evolutionary parameters of this species. Two different demographic models applied to data from seven chloroplast microsatellite loci among 59 accessions show that the effective population size of C. bursa-pastoris is very small indicating a rapid expansion of the species, a result that is in accordance with fossil and historical data. Against this background, analysis of flowering time variation among accessions suggests that ecotypic differentiation in flowering time has occurred recently in the species' history. Finally, our results also indicate that mononucleotide repeat loci in the chloroplast genome can deteriorate in relatively short periods of evolutionary time. [source] Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cellsPARASITE IMMUNOLOGY, Issue 3 2007H. S. GOODRIDGE SUMMARY Modulation of macrophage/dendritic cell (DC) cytokine production by the filarial nematode phosphorylcholine (PC)-containing product, ES-62, is mediated by Toll-like receptor (TLR) 4 and signal transduction depends on the TLR adaptor MyD88. Intriguingly, comparison of TLR4 knock-out (ko) mice with TLR4 mutant C3H/HeJ mice indicates that ES-62 cytokine responses are not dependent on the Pro712 residue of TLR4, which is crucial for the response to bacterial lipopolysaccharide (LPS). Because other immunomodulatory effects of ES-62 have been attributed to PC we have now investigated, using PC conjugated to ovalbumin (PC-Ova), whether PC is responsible for the interaction of ES-62 with TLR4. PC-Ova mimicked the modulation of interleukin (IL)-12 production by ES-62 in a TLR4- and MyD88-dependent manner and as with native ES-62, PC-Ova effects were not dependent on Pro712. Furthermore, both native ES-62 and PC-Ova suppressed Akt phosphorylation, whereas neither altered the activation of p38 or Erk MAP kinases. To rule out any role for the ES-62 protein component, we tested a PC-free recombinant ES-62 (rES-62) generated in the yeast Pichia pastoris. Surprisingly, rES-62 also modulated IL-12 production, but in a TLR4/MyD88-independent manner. Furthermore, rES-62 strongly activated both the p38 and Erk MAP kinases and Akt. However, recent biophysical analysis suggests there are differences in folding/shape between native and rES-62 and hence data obtained with the latter should be treated with caution. Nevertheless, although our study indicates that PC is likely to be primarily responsible for the modulation of cytokine production observed with native ES-62, an immunomodulatory role for the protein component cannot be ruled out. [source] NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers high-level pathogen resistance under greenhouse and field conditionsPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2010Roxana Portieles Summary Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions. [source] Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificitiesPLANT CELL & ENVIRONMENT, Issue 4 2005BARBARA DE CONINCK ABSTRACT The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed. [source] Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coliPROTEIN SCIENCE, Issue 9 2004Jennifer White CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; GPI, glycophosphatidyl inositol; PpDAF, human DAF1,4 expressed in Pichia pastoris, N glycosylated and with an oligohistidine tag; EcDAF, nonglycosylated human DAF 1,4 expressed in Escherichia coli; nDAF, human native glycosylated (GPI-anchored) DAF from erythrocytes; EcDAF-MP, soluble E. coli human DAF linked through a C-terminal cysteine to the myristoylated peptide APT542; PCR, polymerase chain reaction; SCR, short consensus repeat; TCEP, Tris- (2-carboxyethyl) phosphine Abstract Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli -derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. [source] Identification and Functional Characterization of the Delta 6-Fatty Acid Desaturase Gene from Thamnidium elegansTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 1 2007DEPEI WANG ABSTRACT. A cDNA sequence was cloned from the filamentous fungus Thamnidium elegans As3.2806 using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends method (RACE). Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,380 bp, which encodes a 52.4 kDa peptide of 459 amino acids. The designated amino acid sequence has high similarity with that found in fungal delta 6-fatty acid desaturases: it shows three conserved histidine-rich motifs and two hydrophobic domains. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the open reading frame was cloned into the intracellular expression vector pPIC3.5K and the gene was expressed heterologously in Pichia pastoris. Accumulation of ,-linolenic acid to the level of 6.83% in total fatty acid demonstrated that the deduced amino acid sequence possesses of delta 6-fatty acid desaturase activity. [source] Expression, crystallization and preliminary structural analysis of the ectoplasmic region of apical membrane antigen 1 from Plasmodium vivax, a malaria-vaccine candidateACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004Brigitte Vulliez-Le Normand Apical membrane antigen 1 (AMA1), a type 1 transmembrane protein present in the microneme organelles of Plasmodium, is a leading malaria-vaccine candidate. The ectoplasmic region of AMA1 from P. vivax has been expressed in Pichia pastoris and crystallized in two different forms: an orthorhombic form (space group P212121, unit-cell parameters a = 54.1, b = 76.1, c = 103.9,Å) and a monoclinic form (space group C2, unit-cell parameters a = 150.0, b = 53.8, c = 60.3,Å, , = 113.2°). Native data have been collected to 2.0,Å resolution for the orthorhombic form and 1.8,Å for the monoclinic form. A platinum derivative was prepared for the orthorhombic and monoclinic crystals using K2PtCl4 and data were collected at several wavelengths to obtain phases by the MAD technique. A partial model has been built from the electron-density maps of both forms and refinement is in progress. [source] Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastorisACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2000Glenn E. Dale Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor. The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris. The protein was purified to homogeneity and single crystals have been obtained. Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP,phosphoramidon binary complex and the apo enzyme. [source] Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Sachin Wakadkar Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4,M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9,Å resolution at 100,K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P61/P65, with unit-cell parameters a = 90.2, c = 314.5,Å and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure,activity relationship, assembly and thermal stability of Cu,ZnSODs. [source] Crystallization of Saccharomyces cerevisiae,-mannosidase, a cargo protein of the Cvt pathwayACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Yasunori Watanabe Saccharomyces cerevisiae,-mannosidase (Ams1) is a cargo protein that is transported to the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ams1 functions as a resident hydrolase. Ams1 has been overexpressed in the methylotrophic yeast Pichia pastoris, purified and crystallized in two crystal forms. Form I belongs to space group P21, with unit-cell parameters a = 145.7, b = 127.7, c = 164.0,Å, , = 101.5°. Form II belongs to space group I222 or I212121, with unit-cell parameters a = 127.9, b = 163.7, c = 291.5,Å. Diffraction data were collected from these crystals to a resolution of 3.3,Å for form I and of 2.6,Å for form II using synchrotron radiation. [source] Quantitative physiology of Pichia pastoris during glucose-limited high-cell density fed-batch cultivation for recombinant protein productionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Jan Heyland Abstract Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high-cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed-batch cultivations, very high-cell densities were reached (more than 200,gCDW,L,1) resulting in a recombinant protein titer of about 6.5,g,L,1. To investigate the impact of recombinant protein production and high-cell density fermentation on the metabolism of P. pastoris, we used 13C-tracer-based metabolic flux analysis in batch and fed-batch experiments. At a controlled growth rate of 0.12,h,1 in fed-batch experiments an increased TCA cycle flux of 1.1,mmol,g,1,h,1 compared to 0.7,mmol,g,1,h,1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357,368. © 2010 Wiley Periodicals, Inc. [source] Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rateBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Eda Çelik Abstract The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut+) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry-based model containing 102 metabolites and 141 reaction fluxes. Four fed-batch operations with (MS-) and without (M-) sorbitol were performed at three different constant specific growth rates (h,1), and denoted as M-0.03, MS-0.02, MS-0.03, and MS-0.04. Considering the methanol consumption pathway, the M-0.03 and MS-0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS-0.03 to MS-0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ0. Comparing M-0.03 and MS-0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4-fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS-0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS-0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317,329. © 2009 Wiley Periodicals, Inc. [source] Cloning and expression of a Melanocarpus albomyces steryl esterase gene in Pichia pastoris and Trichoderma reeseiBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Hanna Kontkanen Abstract The ste1 gene encoding a steryl esterase was isolated from the thermophilic fungus Melanocarpus albomyces. The gene has one intron, and it encodes a protein consisting of 576 amino acids. The deduced amino acid sequence of the steryl esterase was shown to be related to lipases and other esterases such as carboxylesterases. Formation of mature protein requires post-translational removal of a putative 18-amino-acid signal sequence and a 13-residue propeptide at the N-terminus. The intronless version of the Melanocarpus albomyces ste1 gene was expressed in Pichia pastoris under the inducible AOX1 promoter. The production level was low, and a large proportion of the total activity yield was found to be present intracellularly. However, the fact that steryl esterase activity was produced by P. pastoris cells carrying the expression cassette confirmed that the correct gene had been cloned. The ste1 gene was subsequently expressed in T. reesei under the inducible cbh1 promoter, and a clearly higher production level was obtained. About 60% of the total activity was bound to the fungal mycelium or to solid components of the culture medium, or existed as aggregates. Triton X-100 was successfully used to recover this activity. The heterologous production system in T. reesei provides a means of producing M. albomyces steryl esterase STE1 reliably in large scale for future studies. © 2006 Wiley Periodicals, Inc. [source] Engineering of Pichia pastoris for improved production of antibody fragmentsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Brigitte Gasser Abstract The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor , (MF, leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. © 2006 Wiley Periodicals, Inc. [source] High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicabilityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Lidija Senerovic Abstract A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPACP.rett)of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPACP.rett activity of approximately 40% (3.8 U/mL vs. 2.7U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPACP.rett purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPACP.rett expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPACP.rett. The enzyme wasimmobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPACP.rett performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g dry based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPACP.rett was finally evaluated by studying the kinetically controlled synthesis of ,-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process. © 2005 Wiley Periodicals, Inc. [source] Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChEBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2002Claire O. Weill Abstract In a previous report, Morel and Massoulié showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulié, 2000). They reported that a 5, Bungarus fragment could partially transfer this property to a CAT expression vector. This appeared to offer the possibility of increasing the production of recombinant proteins. In the present paper, we show that insertion of this fragment in the transcribed region, before the polyadenylation site, may have either stimulatory or inhibitory effects, depending on the vector and on the reporter gene. Since the stimulatory effect of Bungarus coding region could not be attached to a small number of discrete motifs, we reasoned that it might result from a general feature of the sequence. Therefore it might be possible to partially transfer this property to the very homologous human AChE (hHAChE) coding sequence by modifications based on synonymous codons, which increased nucleotide identity between the 5, fragment (721 nucleotides) of bBAChE and hHAChE from 71% to 85%. The production of human AChE in transfected COS cells was increased nearly 2-fold with this modified construct, but still remained about 4-fold smaller than that of Bungarus AChE. There was no change in expression level in transformed Pichia pastoris. We thus confirm that coding sequences can strongly influence gene expression, but in a manner that depends on the context and cannot yet be predicted. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 490,497, 2002. [source] Real-time PCR-based determination of gene copy numbers in Pichia pastorisBIOTECHNOLOGY JOURNAL, Issue 4 2010Sandra Abad Abstract Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here. [source] Enantioselective oxidation of 2-hydroxy carboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 3 2010Shuvendu Das Abstract Glycolate oxidase (GO; (S)-2-hydroxyacid oxidase, EC 1.1.3.15) is a flavin mononucleotide (FMN)-dependent enzyme, which catalyzes the oxidation of 2-hydroxy carboxylic acids to the corresponding 2-keto acids. Catalase has been used as cocatalyst to decompose hydrogen peroxide produced in the reaction, thus limiting peroxide-based side reactions and GO deactivation. GO from spinach and catalase T from Saccharomyces cerevisiae previously coexpressed in Pichia pastoris strain NRRL Y-21001, was permeabilized and used for the oxidation of 3-phenyllactic acid, 3-indolelactic acid, 3-chlorolactic acid, 2-hydroxybutanoic acid, and 2-hydroxydecanoic acid to demonstrate high degree of selectivity to the (S)-enantiomers, leaving (R)-isomers intact. The rates of oxidation ranged from 1.3 to 120.0%, relative to the oxidation of lactic acid to pyruvic acid. The best substrates were 3-chlorolactic acid (110%) and 2-hydroxybutanoic acid (120%). Oxidation was carried out with (R)-, (S)-, and (RS)-3-phenyllactic acid, (RS)-lactic acid, and (RS)-2-hydroxybutanoic acid in 500 mL scale to characterize the products and stoichiometry of the reaction. All (RS)- and (S)-2-hydroxy acids produced 2-keto acids at close to the theoretical yield in 1,9 h. (R)-3-Phenyllactic acid was not oxidized over a period of 9 h. Addition of exogenous FMN and catalase were not required for this oxidation using double recombinant Pichia pastoris whole cells. As GO is absolutely specific to (S)-enantiomers, it can be used for resolution of racemic 2-hydroxy acids to (R)-2-hydroxy acids as well as for production of 2-keto acids. This is the first report on the selectivity of a broad range of 2-hydroxy acids by GO. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Optimization of the expression of recombinant human activin A in the yeast Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 2 2010Dale Fredericks Abstract We report a new procedure to express recombinant human activin A using the methanolic yeast, Pichia pastoris. Optimization of culture procedures has involved comprehensive examination of the effects of culture vessel shape, volume of broth in the induction and expression cultures, methanol concentration, culturing temperature, and pH of the expression cultures. After this optimization, as well as modification of the native cleavage sites, a laboratory scale procedure has been established which routinely produced 2,10 mg/L amounts of this vital growth factor in the highly efficient, eukaryotic yeast system. This system avoids the need to produce this protein and similar TGF-, proteins in mammalian cell lines which, in addition to being costly, produce many native binding partners of these cystine knot proteins, a factor which can dramatically affect yields of the target protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] | ||||||||||||||||||||||||||||||||||