研究了基因工程菌P ich ia p astotis高密度、高表达的培养条件。在全合成摇瓶培养基的基础上,考察了诱导过程中甲醇浓度、温度、pH、(NH4)2SO4及磷酸盐浓度等因素对猪胰岛素前体(P IP)表达的影响。研究表明:甲醇的最佳诱导浓度为6 g/L...研究了基因工程菌P ich ia p astotis高密度、高表达的培养条件。在全合成摇瓶培养基的基础上,考察了诱导过程中甲醇浓度、温度、pH、(NH4)2SO4及磷酸盐浓度等因素对猪胰岛素前体(P IP)表达的影响。研究表明:甲醇的最佳诱导浓度为6 g/L,过高或过低的甲醇浓度都不利于菌体生长和蛋白表达;在菌体生长和蛋白表达阶段,pH应分别控制在5.5和4.0;诱导阶段,培养温度下降到28°C,可以提高重组蛋白质的表达量。上述结果用于15 L全自动发酵罐实验,P IP表达水平达到了1.69 g/L,是摇瓶结果的17倍。展开更多
The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rA...The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.展开更多
Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragment...Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.展开更多
A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotid...A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.展开更多
Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sampl...Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.展开更多
The plasmid pTLIL-2,which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E.coli,was reconstructed:a series of deletions were made in 3'non-coding region of human IL-2 cDNA,and 7 recombin...The plasmid pTLIL-2,which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E.coli,was reconstructed:a series of deletions were made in 3'non-coding region of human IL-2 cDNA,and 7 recombinant plasmids with different deletions were selected,on the other hand, a Tac promoter sequence from pDR540 was inserted to upstream of IL-2 cDNA in pTLIL-2 so that pTLIL-2DT,which contains double Tac promoters,was constructed.And then,E.coli JM103 was transformed with the above 8 recombinant plasmids respectively.The expression efficiency of IL-2 cDNA in E.cbli transformed with different plasmids was evaluated by means of SDS-PAGE and measuring 3H-TdR incorporation of IL-2-dependent activated T lymphocytes in the presence of bacterial extracts.Three engineering strains with high efficiency of IL-2 expression were selected,and all of these strains could produce recombinant IL-2(rIL-2)4 times more than E.coil JM103/pTLIL-2.展开更多
Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification...Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.展开更多
Five new-type Fc receptor molecules were constructed based on streptococcal protein G (SpG) and staphylococcal protein A (SpA). These protein molecules contain one to six Fc binding domains to immunoglobulins which ar...Five new-type Fc receptor molecules were constructed based on streptococcal protein G (SpG) and staphylococcal protein A (SpA). These protein molecules contain one to six Fc binding domains to immunoglobulins which are structurally different from native SpG or SpA. Their expression levels reached 17-30% of the total bacterial proteins after heat induction in E. colt. Immunodiffusion and ELISA results showed that the engineered protein TG (184 amino acid residues) composed of three SpG C3 domain could bind more broadly and efficiently than the native SpG to the IgGs of human, goat, rabbit, etc. , and its optimal pH for binding became wider (pH5-8) compared with the SpG (pH5) ; and the protein TGA (357AA), fused by protein TG and the A, B, C domains of SpA, displayed both the binding pattern of SpG and SpA.展开更多
文摘研究了基因工程菌P ich ia p astotis高密度、高表达的培养条件。在全合成摇瓶培养基的基础上,考察了诱导过程中甲醇浓度、温度、pH、(NH4)2SO4及磷酸盐浓度等因素对猪胰岛素前体(P IP)表达的影响。研究表明:甲醇的最佳诱导浓度为6 g/L,过高或过低的甲醇浓度都不利于菌体生长和蛋白表达;在菌体生长和蛋白表达阶段,pH应分别控制在5.5和4.0;诱导阶段,培养温度下降到28°C,可以提高重组蛋白质的表达量。上述结果用于15 L全自动发酵罐实验,P IP表达水平达到了1.69 g/L,是摇瓶结果的17倍。
基金supported by a grant from the Public Benefit Research Foundation of China (200903052)the Science and Technology Department of Guangdong Province, China (2009A020101003)
文摘The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.
基金Supported by Natural Science Foundation of Yunnan Province(2011FB049)National Natural Science Foundation of China(31260481,31460516)+2 种基金Fund of Yunnan Education Department(2013Y251)Fund of the Department of Life Science and Technology,Kunming University(GXKM201505)Talent Fund for PhD(YJL11015)
文摘Using genomic DNA of bolting-tolerant lettuce as a template,flanking fragments of lettuce plastid rpo A gene were amplified and cloned by PCR. Targeting the sites of these two fragments,homologous recombinant fragments of exogenous gene were integrated to construct lettuce plastid expression vector p Brpo AGFP,which harbored the expression cassette Prrn-gfp-aad A-Tpsb A. The results showed that the amplified flanking fragments were 1.2 and 1.1 kb in size. After sequencing,restriction digestion,ligation and transformation,lettuce plastid expression vector containing expression cassette Prrn-gfp-aad A-Tpsb A was constructed and confirmed by SDS-PAGE electrophoresis. The results of SDS-PAGE electrophoresis indicated that gfp gene was efficiently expressed under the regulation of plasmid specific promoter Prrn and terminator Tpsb A. GFP accounted for 45. 6% of total soluble proteins; inclusion bodies accounted for 47.5 % of bacterial proteins,which reached relatively high expression levels. The construction of lettuce plastid expression vector p Brpo A-GFP laid a solid foundation for establishment of subsequent lettuce plastid transformation system and genetic improvement of lettuce using various functional genes.
基金Supported by Prime Minister FouNdationthe High Techriology Research and Development Programme of ChinaRockefeller Foun-dation.
文摘A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.
文摘Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.
文摘The plasmid pTLIL-2,which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E.coli,was reconstructed:a series of deletions were made in 3'non-coding region of human IL-2 cDNA,and 7 recombinant plasmids with different deletions were selected,on the other hand, a Tac promoter sequence from pDR540 was inserted to upstream of IL-2 cDNA in pTLIL-2 so that pTLIL-2DT,which contains double Tac promoters,was constructed.And then,E.coli JM103 was transformed with the above 8 recombinant plasmids respectively.The expression efficiency of IL-2 cDNA in E.cbli transformed with different plasmids was evaluated by means of SDS-PAGE and measuring 3H-TdR incorporation of IL-2-dependent activated T lymphocytes in the presence of bacterial extracts.Three engineering strains with high efficiency of IL-2 expression were selected,and all of these strains could produce recombinant IL-2(rIL-2)4 times more than E.coil JM103/pTLIL-2.
文摘Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.
文摘Five new-type Fc receptor molecules were constructed based on streptococcal protein G (SpG) and staphylococcal protein A (SpA). These protein molecules contain one to six Fc binding domains to immunoglobulins which are structurally different from native SpG or SpA. Their expression levels reached 17-30% of the total bacterial proteins after heat induction in E. colt. Immunodiffusion and ELISA results showed that the engineered protein TG (184 amino acid residues) composed of three SpG C3 domain could bind more broadly and efficiently than the native SpG to the IgGs of human, goat, rabbit, etc. , and its optimal pH for binding became wider (pH5-8) compared with the SpG (pH5) ; and the protein TGA (357AA), fused by protein TG and the A, B, C domains of SpA, displayed both the binding pattern of SpG and SpA.
