AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and ...AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.展开更多
The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A, under the control of the AOX1 promoter. The recombi...The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A, under the control of the AOX1 promoter. The recombinant plasmid pHBM905A-85B linearized by Sal I was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35 × 10^3 approximately detected by SDS-PAGE and Western blot. EI,ISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinant M. tuberculosis Ag85B in P. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.展开更多
Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide ...Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.展开更多
利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗...利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗性酵母菌株 ,甲醇诱导目的蛋白表达。经 SDS- PAGE和 Western blot检测表明 ,Bo IL- 2以融合蛋白形式在胞内表达 ,但没能分泌到胞外。通过 Bo IL- 2在巴斯德毕赤酵母中的表达 ,重点讨论了信号肽。展开更多
基金Supported by the grants from National High Technology ResearchDevelopment Program, No. 2002AA2Z345BNo. 2004AA2Z3803 of the Ministry of Science and Technology of China
文摘AIM： To study the secretory expression of human hepatocyte growth factor （hdHGF） gene in Pichia pastoris. METHODS： The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coilyeast shuttle vector of pPIC9. The constructed plasmid, pPIC9-hdHGF, was transformed into the GSl15 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Hut ＋ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor. RESULTS： The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g （DCW）/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION： The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.
文摘The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A, under the control of the AOX1 promoter. The recombinant plasmid pHBM905A-85B linearized by Sal I was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35 × 10^3 approximately detected by SDS-PAGE and Western blot. EI,ISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinant M. tuberculosis Ag85B in P. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.
基金Supported by the National Natural Science Foundation of China (No. 30200294)
文摘Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.
文摘利用含有强启动子 PAOX1 和 α-因子信号肽序列的巴斯德毕赤酵母载体 p PICZαA,构建出含牛白细胞介素 2(Bo IL - 2 )基因的重组质粒 Bo IL 2 - p PICZαA。线性化的重组表达载体转化到巴斯德毕赤酵母 X- 33及 KM71H中 ,筛选Zeocin高抗性酵母菌株 ,甲醇诱导目的蛋白表达。经 SDS- PAGE和 Western blot检测表明 ,Bo IL- 2以融合蛋白形式在胞内表达 ,但没能分泌到胞外。通过 Bo IL- 2在巴斯德毕赤酵母中的表达 ,重点讨论了信号肽。
