Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Exendin-4, a peptide analogue of glucagon-like peptide-1 (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 fusion protein, expressed by methanol induction in Pichia pastoris system, was purified to homogeneity by chromatography followed by enterokinase cleavage of the fusion protein and subsequent purification of the recombinant exendin-4. Purity of the recombinant exendin-4 was 95.6%. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.     

Expression of Bacterial Hemoglobin in the Yeast, <Emphasis Type="Italic">Pichia pastoris,</Emphasis> with a Low O<Subscript>2</Subscript>-Induced Promoter

The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O₂ tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O₂-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds.     

High expression of Trigonopsis variabilis -amino acid oxidase in Pichia pastoris

To explore a new approach of high expression of -amino acid oxidase (DAAO) in Pichia pastoris, a gene encoding DAAO from Trigonopsis variabilis (TvDAAO gene) deleted intron was prepared by PCR amplification and cloned into the intracellular expression vector pPIC3.5K. The expression plasmid pPIC3.5K-DAAO linearized by SalI was transformed into Pichia pastoris strain GS115 (his⁻mut⁺). By means of MM and MD plates and PCR, the recombinant P. pastoris strains (his⁺mut⁺) were obtained. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant strain PD27 with the highest expression of DAAO was screened through activity assay and its high-density fermentation was carried out in a 1-l fermentor. Activity assay and SDS-PAGE demonstrated that DAAO was intracellularly expressed in P. pastoris with the induction of methanol. The recombinant cells with high expression of DAAO were screened and the high-density fermentation was carried out in a 1-l fermentor. Interestingly, the DAAO expression level reached up to 473 U/g dry cell weight in fermentation yield. Finally, 1-hexanol was used to break recombinant cells and the specific activity of DAAO was 1.46 U/mg protein in crude extraction.     

卡氏德巴利酵母植酸酶在毕赤酵母中的异源表达及优化

【背景】植酸是一种能螯合金属离子和蛋白质的有机磷类化合物,广泛存在于植物组织中,影响动物对营养元素的吸收。在饲料中加入植酸酶可有效降解植酸。【目的】构建毕赤酵母异源表达卡氏德巴利酵母(Debaryomyces castellii,D. castellii)植酸酶的菌株,促进卡氏德巴利酵母植酸酶的研究及工业应用。【方法】将卡氏德巴利酵母植酸酶基因进行密码子优化后转入毕赤酵母GS115中,通过筛选多拷贝、敲除蛋白酶、过表达分子伴侣及转运蛋白的方法获取优势菌株。【结果】所得重组菌株GS115/DCphy(ΔPep4)(BFR2)的产酶酶活是低拷贝菌株的7倍。【结论】研究结果为卡氏德巴利酵母植酸酶的异源表达及潜在工业应用提供了一定的指导。     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

High-level secretory expression, purification and characterization of Ailuropoda melanoleuca growth hormone in Pichia pastoris

Growth hormone is one of the most important hormones, which is involved in many reproductive processes of giant panda Ailuropoda melanoleuca. In this study, the mature peptide of A. melanoleuca growth hormone (AmGH) was successfully expressed and secreted in Pichia pastoris under the control of AOX1 promoter. The expression condition for AmGH in P. pastoris, such as the expression time, pH value and methanol concentration in the BMMY were optimized and the AmGH expression level is about 100 mg/L using GS115 recombinant under optimized condition (96 h of 1.5% methanol induction). The secreted nascent AmGH were purified using ammonium sulfate fractionation. The mature AmGH protein exhibited a molecular mass of approximately 22 kDa on SDS–PAGE. This study would provide a new opportunity for large-scale expression and purification of AmGH, which might facilitate studies on the biological activity of AmGH.     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l^–1 at the 200 l scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.     

Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris

Cellular targeting of lycopene biosynthetic enzymes was investigated in Pichia pastoris X-33. Three lycopene pathway enzymes, CrtE, CrtB, and CrtI, were fused to fluorescent EGFPs with or without a peroxisomal targeting sequence (PTS1) and then expressed in P. pastoris. When P. pastoris was grown in YPD, the PTS1 fusion enzymes were found to be localized in peroxisomes, whereas the enzymes not fused with PTS1 were equally distributed throughout the entire cell. A similar targeting pattern was also observed in P. pastoris strains that were grown in peroxisome-proliferating medium, YPOT. Analysis of the fluorescent images of isolated peroxisomes showed that the PTS1 fused enzymes were dominantly present in peroxisomes whereas small amount of the enzymes not fused with PTS1 were non-specifically sent to peroxisomes. These results indicate that PTS1 specifically target lycopene pathway enzymes into peroxisomes and this targeting pathway was strong enough to overcome their inherent targeting program. In conclusion, we first showed that carotenogenic enzymes can be targeted into the specific cellular location of recombinant hosts and this targeting strategy can serve as the basis for the subsequent development of sophisticated pathway engineering in microorganisms.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.     

Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25–50% less active (p<0.05) at pH 2 to 3.5 or at 45 and 55 °C, but 50% more active (p<0.05) at 75 °C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p<0.05) than that of the glycosylated phytase at 45 and 55 °C, but was 80 and 94% lower (p<0.05) at 65 ° and 75 °C, respectively.     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

过表达PDI对毕赤酵母分泌淀粉样蛋白的影响——以胱抑素为例

[目的] 本研究旨在结合酵母菌蛋白质二硫键异构酶（protein disulfide isomerase,PDI）与其底物蛋白鸡胱抑素C （chicken cystatin C,cC）在酵母中的共表达,理解PDI影响外源蛋白合成与表达的调控规律。运用转录组深度测序技术（RNA-Seq）筛选差异基因,调取并鉴定影响cC表达的关键基因,为解析外源蛋白高效表达机制,改造工程菌株提供理论支撑。[方法] 以巴斯德毕赤酵母GS115、GS115-cC为出发菌株,采用电转的方法将携带PDI编码基因的载体pPIC3.5K转入到GS115/GS115-cC菌株,使其在菌株中过表达,研究过表达PDI对cC表达的影响。采用RNA-Seq深度测序方法,研究重组毕赤酵母基因表达差异情况。并结合KEGG注释结果对数据进行分析,挑选差异显著表达基因进行验证,初步明确其在蛋白表达调控方面的功能。[结果] 本研究通过构建过表达PDI重组毕赤酵母菌株,使得外源蛋白cC的表达量显著增加。利用RNA-seq技术分析过表达PDI菌株与正常菌株的差异,最终筛选了373个差异表达基因,其中有122个差异基因注释到KEGG生物通路,包括12个基因注释到蛋白质转运和分解代谢途径,21个基因注释到蛋白质折叠分选和降解途径,以及24个基因参与蛋白质的翻译途径等。[结论] 在毕赤酵母中过表达PDI能显著增加外源蛋白cC的表达量。通过对过表达与正常表达PDI的毕赤酵母基因的表达谱分析,初步确定了其中一些转录情况变化显著的基因,明确了它们参与的细胞途径和信号通路,为改造具有高效率表达淀粉样蛋白的酵母菌株奠定基础。     

Expression,purification, and characterization of recombinant Chinese shrimp crustin-like protein (CruFc) in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A crustin-like protein (CruFc) from Fenneropenaeus chinensis was expressed in Pichia pastoris and then purified to electrophoretic homogeneity on a Sephacryl S-100 column with a band corresponding to the expected one (13 kDa) shown by 15% SDS-PAGE. Western blot indicated that the rCruFc specifically reacted with polyclonal rabbit anti-Fenneropenaeus chinensis CruFc. Production in a 5 l bioreactor gave 237 mg rCruFc/l. Antimicrobial assay revealed that 4 μM rCruFc inhibited growth of Staphylococcus aureus.     

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His₆-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.     

Construction,production, and characterization of recombinant scFv antibodies against methamidophos expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos for downstream applications.     

Overexpression of an optimized <Emphasis Type="Italic">Aspergillus sulphureus β</Emphasis>-mannanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris.     

High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.