Objective To try making huZP3a22-176 and huZP3b177-348 polypeptides (representing anintact huZP322-348 protein without its N-terminal signal peptide and C-terminal trans-membrane domain ) express in E. coli at a highe...Objective To try making huZP3a22-176 and huZP3b177-348 polypeptides (representing anintact huZP322-348 protein without its N-terminal signal peptide and C-terminal trans-membrane domain ) express in E. coli at a higher levelMethods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method.The pBV221 plasmid was used to construct thermo-inducible recombinant expressionvector. Purification of two target expression products employed an improved methodof preparative gel polyacrylamide gel electrophoresis.Results Two polypeptides of recombinant huZP3a (rhuZP3a) and recombinant huZP3b(rhuZP3b) were all expressed respectively in an E. coli BL21(DE3)pLysS strain at ahigher level, which were recognized by two specific polyclonal antisera in Westernblotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3brespectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3bwith more than 95% relative homogeneity were harvested from 1 L culture respectively.Conclusion The availability of two rhuZP3 polypeptides will help in detecting theimmunogenicities of rhuZP3a and rhuZP3b through animal experiments andconfirming the function domain of non-glycosylated huZP3 to induce acrosomereaction in vitro.展开更多
目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码...目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码子.将获得的LZP 3突变基因(LZP 3m)插入pGAPZαA中,构建穿梭表达载体.以重组体转化P ich ia Pastoris SM D 1168菌株进行表达.结果:LZP 3m基因的表达量比野生型LZP 3基因明显提高.结论:通过密码子优化,能显著提高LZP 3基因在酵母细胞中的表达水平.展开更多
基金This work was supported by grant (No. 03JG05014) from the Population Family Planning Commission ofShanghai. China and the Medical and Health Science Research Foundation of Zhejiang Province(No. 2004A002)
文摘Objective To try making huZP3a22-176 and huZP3b177-348 polypeptides (representing anintact huZP322-348 protein without its N-terminal signal peptide and C-terminal trans-membrane domain ) express in E. coli at a higher levelMethods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method.The pBV221 plasmid was used to construct thermo-inducible recombinant expressionvector. Purification of two target expression products employed an improved methodof preparative gel polyacrylamide gel electrophoresis.Results Two polypeptides of recombinant huZP3a (rhuZP3a) and recombinant huZP3b(rhuZP3b) were all expressed respectively in an E. coli BL21(DE3)pLysS strain at ahigher level, which were recognized by two specific polyclonal antisera in Westernblotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3brespectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3bwith more than 95% relative homogeneity were harvested from 1 L culture respectively.Conclusion The availability of two rhuZP3 polypeptides will help in detecting theimmunogenicities of rhuZP3a and rhuZP3b through animal experiments andconfirming the function domain of non-glycosylated huZP3 to induce acrosomereaction in vitro.
基金This work was supported by Shanghai Municipal Population and Family Planning Commission Foundation(No.03JG05014)Medical and Health Science Research Foundation of Zhejiang Province(No.2004A002).
文摘目的:提高草原兔尾鼠(L agurus lagurus)卵透明带3(LZP 3)基因在酵母细胞中表达的水平.方法:利用重叠PCR技术,定点突变LZP 3基因上6个稀有的密码子簇,将LZP 3基因中11个稀有的密码子更换成毕赤酵母(P ich ia Pastoris)最常用的相应密码子.将获得的LZP 3突变基因(LZP 3m)插入pGAPZαA中,构建穿梭表达载体.以重组体转化P ich ia Pastoris SM D 1168菌株进行表达.结果:LZP 3m基因的表达量比野生型LZP 3基因明显提高.结论:通过密码子优化,能显著提高LZP 3基因在酵母细胞中的表达水平.
