Secretory expression of insulin precursor in pichia pastoris and simple procedure for producing recombinant human insulin

In this work, Pichia pastoris was applied to produce human insulin by a simple procedure. The synthesized insulin precursor (ILP) gene was inserted into pPIC9K to obtain secretary expression plasmid pPIC9K/ILP. Pichia pastoris GS115 was transformed by pPIC9K/ILP and the high expresser was screened. In a 16 L fermentor, the insulin precursor production was 3.6 g/L. Insulin precursor, purified by one-step chromatography, was converted into human insulin by transpeptidation. The yield of the processing procedure from insulin precursor to insulin reached up to 70%. In vivo assay showed that the biological activity of the produced recombinant human insulin was 28.8 U/mg.     

重组人血管内皮细胞生长因子121 cDNA的基因克隆、表达和鉴定

应用RT-PCR方法,扩增人VEGF₁₂₁ cDNA基因片段,与酵母表达载体pPIC9K重组,获得表达质粒p9KVEGF₁₂₁.该质粒转化毕赤酵母菌GS115,用G418-YPD平板筛选高拷贝转化子,PCR鉴定VEGF₁₂₁ cDNA与酵母染色体整合状态,高拷贝转化子用甲醇诱导表达.工程菌用5 L发酵罐发酵,表达产物r-hVEGF₁₂₁占培养液中总蛋白量70%以上.纯化产物促进牛毛细血管内皮(BCE)细胞增殖,并强烈促进血管通透.     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

牛流行热病毒糖蛋白G1抗原表位在毕赤酵母中的表达和抗原性鉴定

从包含牛流行热病毒G蛋白基因的质粒pMD-G中克隆G₁抗原表位区基因,亚克隆进表达载体pPIC9K,构建重组载体pPIC9K-G₁,线性化后电转化毕赤酵母GS115,通过G418压力和PCR法筛选阳性重组酵母进行诱导表达。经SDS-PAGE、脱糖基化分析、Western blot、ELISA、兔体免疫实验和特异性分析,表明该基因在GS115中表达并进行了适度的糖基化,表达蛋白有良好的生物学活性和特异性,可作为包被抗原,开发ELISA诊断试剂盒。     

Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris

The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K _cat /K _m of recombinant OPH was 598 mM^?1 s^?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.     

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED₅₀ of 0.5 mg/kg.     

Reduced methanol input induces increased protein output by <Emphasis Type="Italic">AOX1</Emphasis> promoter in a <Emphasis Type="Italic">trans</Emphasis>-acting elements engineered <Emphasis Type="Italic">Pichia pastoris</Emphasis>

High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69 g/(L supernatant) at the methanol feeding rate of 6.67 mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12 mL/(h·L broth). Correspondingly, the insulin precursor expression level in fermentation broth and maximum specific insulin precursor production rate was 137 and 77% higher than the WT-IP, respectively. However, oxygen consumption and heat evolution were reduced, and the highest oxygen consumption rate and heat evolution rate of the MF1-IP were 18.0 and 37.7% lower than the WT-IP, respectively.     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification, and bioactivity analysis of the expressed products were performed. __________ Translated from Microbiology, 2006, 33(1): 1–6 [译自: 微生物学通报]     

Overproduction of a truncated poly (vinyl alcohol) dehydrogenase in recombinant Pichia pastoris by low-temperature induction strategy and related mechanism analysis

Based on the N-terminal sequencing of poly (vinyl alcohol) dehydrogenase (PVADH), a 1,644-bp gene encoding a truncated PVADH (tPVADH) was amplified using the synthetic gene (GenBank accession No. JQ235753) as a template, and was further transformed into Pichia pastoris GS115 with the vector pPIC9K. The maximal tPVADH activity reached 546 U/mL in shake flask. The influence of methanol concentration and induction temperature on tPVADH production was further investigated in 3-L bioreactor. When the methanol concentration and induction temperature were controlled at 15 g/L and 22 °C, respectively, the maximal tPVADH activity reached 8,464 U/mL, which was nearly 10 times that of mature PVADH expressed under the same condition and was the highest level ever reported. The reason responsible for the significant improvement of tPVADH production at low induction temperature was explored in terms of cell viability, extracellular proteases activity, and alcohol oxidase activity.     

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K^M vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K^M-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn²⁺, which caused a strong inhibition. The K _m and V _max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.     

Expression and antigenic characterization of the epitope-G1 of the Bovine ephemeral fever virus glycoprotein in Pichia pastoris

The epitope-G₁ gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G₁. Then the pPIC9K-G₁ was linearized and transformed into Pichia pastoris GS115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G₁ was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G₁ protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis. Foundation item: National Dairy Foundation of China (2002BA518A04)     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris

A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris. The fragment was then amplified by polymerase chain reaction and inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using SalI and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca²⁺ and Mg²⁺ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant (K _m) of 1.89 mg/mL and a maximum reaction rate (V _max) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris, and the achieved PVADH yield is the highest ever reported.     

High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris

The porcine circovirus type 2 (PCV2) capsid protein (Cap) is an important antigen for the development of vaccines. To achieve high-level expression of recombinant PCV2 Cap in Pichia pastoris, the wild-type Cap (wt-Cap) and optimized Cap (opti-Cap) gene fragments encoding the same amino acid sequence of PCV2 were amplified by PCR using DNA from lymph nodes of postweaning multisystemic wasting syndrome-suffered pigs and synthesized based on the codon bias of the methylotrophic yeast P. pastoris, respectively. The wt-Cap and opti-Cap gene fragments were inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the alcohol oxidase 1 (AOX1) promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmids, designated as pPIC9K-wt-Cap and pPIC9K-opti-Cap, were linearized using SacI and transformed into P. pastoris GS115 by electroporation. The expressed intracellular soluble opti-Cap reached 174 μg/mL without concentration in a shake flask and kept good reactivity to PCV2-specific positive sera, whereas the wt-Cap could not be detectable throughout three times electroporation. Strong specific PCV2-Cap antibodies were elicited from piglets immunized with vaccine based on opti-Cap. To the best of our knowledge, the achieved opti-Cap yield is the highest ever reported. Our results demonstrated that codon optimization play an important role on the high-level expression of a codon-optimized PCV2-Cap gene in P. pastoris, and the vaccine based on opti-Cap may be a potential subunit vaccine candidate.     

Cloning and expression of defibrase cDNA from the venom of Gloydius shedaoensis * *

The total RNA was extracted from the venom gland of snake, Gloydius shedaoensis. The defibrase cDNA was amplified by RT-PCR. Assaying the nucleotide sequence of the cDNA allowed the complete amino acid sequence for defibrase of Gloydius shedaoensis to be determined. The defibrase cDNA was inserted into the vector, pPIC 9K, and expressed in Pichia pastoris which then produced 10 mg target protein l^–1.     

Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.     

Heterologous production of the stain solving peptidase PPP1 from <Emphasis Type="Italic">Pleurotus pulmonarius</Emphasis>

A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.     

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris

Human α-defensin 5 (HD₅), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD₅ through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD₅ mature peptide (rmHD₅) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD₅ by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD₅ into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD₅ vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD₅ nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD₅, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD₅. The results showed that about 165.0 mg/l of rmHD₅ with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD₅ was recovered after purification. The in vitro experiments revealed that rmHD₅ exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD₅ in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.     

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.     

A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 (III) chain in Pichia pastoris

Recombinant human full-length mature collagen α1 (III) chain (rhCOL3A1) was secreted by Pichia pastoris GS115, using the Saccharmyces cerevisiae á-mating factor prepro signal, and the theoretical molecular weight of rhCOL3A1 was 95.344 kDa. The gene cloned from human placenta, was designed and cloned into expression vector pPIC9K under the control of a strong inducible promoter AOX1.The expression stage of rhCOL3A1 was sensitive to different carbon ratios through mixed fermentation. LCMS/ MS analysis and western blotting demonstrated that the recombinant human full-length mature collagen a1 (III) gene was successfully expressed in P. pastoris GS115 during the methanol induction stage. Furthermore, an effective strategy of mixed fermentation was established to express rhCOL3A1 in shake flash. Compared to single carbon induction, when induced with mixed carbon at the ration of 0.8 (glycerol/methanol), the time corresponding to the highest yield of rhCOL3A1 (1.27 g/L) was drastically reduced by 50%. The same conclusion was observed from RT-qPCR. Consequently, a new strategy which was more time-saving and effective was provided for the large-scale producing the full-length mature rhCOL3A1.