Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.     

Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris

A 371 bp full-length cDNA (GenBank Accession No. DQ232774) was obtained from housefly Musca domestica by using degenerate primers and subsequent amplification by 5'- and 3'-RACE. The cecropin gene, Mdcec and Mdcec/6His, was cloned into expression pPICZalpha-A vector and was expressed in the methylotrophic yeast, Pichia pastoris. The recombinant Mdcec was purified using cationic exchange chromatography and 1.2mg pure active Mdcec was obtained from 100ml culture broth supernatant. To facilitate purification of Mdcec, the C-terminal 6His-tagged Mdcec was also expressed in P. pastoris. The recombinant Mdcec/6His was purified to homogeneity by a nickel chelating sepharose column and 2.0mg pure active Mdcec/6His was obtained from 100ml culture broth supernatant. Anti-microbial assays demonstrated that Mdcec had broad spectrum of antimicrobial property against fungi, as well as Gram-positive and Gram-negative bacteria. Mdcec/6His showed a similar activity to Mdcec against bacteria, but a slight higher activity against fungi. These results indicate that the 6His-tag can enhance the cationic nature and stability of Mdcec. This is the first report on the heterologous expression of a cecropin and cecropin with a 6His tag in P. pastoris. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional M. domestica cecropin for both research and industrial purpose.     

Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris

Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ～14 kD, as shown by SDS-PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni-NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M.lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.     

Expression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris

The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.     

Expression and characterization of human tumor necrosis factor-related apoptosis inducing ligand in methylotrophic yeast Pichia pastoris

Recombinant human TRAIL was successfully expressed in a secreted form in methyltrophic yeast Pichia pastoris induced by methanol. The expressive product was immunoreactive to TRAIL antibody. The addition of the expressive product into cultured human lung cancer cell A549 showed moderate cell death, typical morphological characterization of apoptotic bodies, and DNA fragmentation. This result provided a new method to produce recombinant human TRAIL as a cancer therapeutic drug.     

Production and application of methylotrophic yeast Pichia pastoris

Pichia pastoris is a methylotrophic yeast that makes use bf the enzyme alcohol oxidase to catalyze the first step of the dissimilatory pathway that enables it to grow on methanol. Because of its stability and low substrate specificity, alcohol oxidase is of considerable interest for a range of biotechnological processes. Various feeding regimes were evaluated in an effort to increase the biomass concentration and productivity that could be achieved from fermentations using this organism. Through continuous or semicontinuous feeding, biomass concentrations were increased 10-fold over those achieved in batch fermentations. In subsequent trials, nongrowing whole cells were applied successfully to convert ethanol to acetaldehyde. Quantitative conversions of 20-g/L solutions of ethanol have been achieved in 2 h, and acetaldehyde concentrations of up to 35 g/L have been achieved using extended reaction times of 5 h. The conversion reaction was limited by end product inhibition and by acetaldehyde holdup within the yeast cells.     

Expression and characterization of the hepatitis E virus ORF3 protein in the methylotrophic yeast,Pichia pastoris

We have used the methylotrophic yeast, Pichia pastoris, to express the open reading frame 3 (ORF3) of the hepatitis E virus (HEV). The ORF3 gene codes for a 123-amino-acid protein that contains highly immunodominant epitopes and is a potentially useful diagnostic and immunoprophylactic antigen. The expressed protein showed positive on immunoblots probed against antibodies raised in rabbit and infected human patient sera. In order to optimize the ORF3 protein expression, we have examined the regulated expression of this protein and characterized it. Unlike its expression in E. coli, the ORF3 protein was present in both the soluble and insoluble fractions of the cell lysate. The expressed protein is not glycosylated and does not undergo any major processing in the host strain.     

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of P. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.     

Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris

The extracellular beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-beta-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-beta-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.     

Expression of biologically active recombinant pokeweed antiviral protein in methylotrophic yeast Pichia pastoris

Pokeweed antiviral protein (PAP)-I from the spring leaves of Phytolacca americana is a naturally occurring RNA-depurinating enzyme with broad-spectrum antiviral activity. Interest in PAP is growing due to its use as a potential anti-HIV agent. However, the clinical use of native PAP is limited due to inherent difficulties in obtaining sufficient quantities of homogeneously pure active PAP without batch-to-batch variation from its natural resource. Here, we report the expression of mature PAP (residues 23 to 284) with a C-terminal hexahistidine tag in the methylotrophic yeast Pichia pastoris, as a secreted soluble protein. The final yield of the secreted PAP is greater than 10 mg/L culture in shaker flasks. The secreted recombinant protein is not toxic to the yeast cells and has an apparent molecular mass of 33-kDa on SDS-PAGE gels. The in vitro enzymatic activity and cellular anti-HIV activity of recombinant PAP were of the same magnitude as those of the native PAP purified from P. americana. To our knowledge, this is the first large-scale expression and purification of soluble and biologically active recombinant mature PAP from yeast.     

Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.     

抗菌肽VIP在毕赤酵母中的高效表达及鉴定

基于人抗菌肽VIP(Vasoactive intestinal peptide)基因序列,按照毕赤酵母密码子偏好性设计引物;用SOE-PCR法扩增目的基因;然后将目的基因克隆至毕赤酵母分泌型表达载体pPICZαA上,构建VIP分泌表达菌株GS115-p PICZαA-vip。用甲醇诱导96 h收集上清,用质谱进行鉴定,结果显示分泌表达产物与人抗菌肽VIP理论值(3 326.82 Da)完全一致,表明人抗菌肽VIP成功得到分泌表达。琼脂糖凝胶扩散法实验结果显示,重组VIP对大肠杆菌Escherichia coli ATCC25922和金黄色葡萄球菌Staphylococcus aureus ATCC25923都有很强的抗菌活性,MIC(Minimal inhibitory concentration)分别为8 mmol/L和16 mmol/L。进一步细胞毒性和溶血性实验结果显示,重组VIP对正常细胞NCM460和IPEC-J2没有毒性,其对SD大鼠红细胞不具有溶血活性。通过透射电镜观察了VIP的抗菌机制,结果显示VIP主要通过破坏细胞膜的方式抑杀细菌。本研究为人抗菌肽VIP的开发应用和大量生产奠定了基础。     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B₂ receptor (B₂R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast Pichia pastoris. In the expression vector, B₂R gene was driven under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and Western blot analysis, it was proved that B₂R recombinant receptor proteins were expressed at a high level in the yeast. Furthermore, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B₂R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy. The text was submitted by the authors in English.     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B2 receptor (B2R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast of Pichia pastoris. In the expression vector, B2R gene was drove under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and western blot analysis, it was proved that B2R recombinant receptor proteins were expressed at high level in the yeast. Further more, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B2R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy.     

Production and characterization of recombinant Phanerochaete chrysosporium cellobiose dehydrogenase in the methylotrophic yeast Pichia pastoris.

The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.     

Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

Summary Alcohol oxidase biosynthesis was induced when Pichia pastoris was grown in a medium containing methanol as the sole carbon and energy source. Specific activity was highest during the logarithmic phase of growth (1.22 g acetaldehyde produced/g cell dry wt. per hour), and declined steadily thereafter. The addition of 0.1% (w/v) yeast extract to the methanol growth medium promoted higher biomass production, increased alcohol oxidase specific activity, and contributed to increased enzyme stability under use conditions. When P. pastoris was used for wholecell bioconversions, 30.2 g of ethanol were oxidized to 28 g acetaldehyde in 12 h, at a carbon recovery of 97%. Acetaldehyde concentrations in excess of 1 M were achieved when the concentration of the TRIS buffer, used to chemically trap the acetaldehyde, was increased to 1 M.Issued as NRCC no. 30256Offprint requests to: W. D. Murray     

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.     

Modification of the protein glycosylation pathway in the methylotrophic yeast Pichia pastoris

-1,2-Mannosidase from Trichoderma reesei was used to modify the N-linked glycosylation pathway of the methylotrophic yeast Pichia pastoris. Expression of foreign influenza glycoproteins with more extensively processed N-linked oligosaccharides was observed when -1,2-mannosidase was secreted in the culture medium. However, intracellular removal of mannose residues may stimulate mannosyltransferase activity and leads to hyperglycosylation. [3H]Mannose suicide selection or high concentrations of orthovanadate, commonly used to isolate glycosylation mutants of Saccharomyces cerevisiae, had no profound effect on Pichia pastoris. © Rapid Science Ltd. 1998     

Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization

S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.