摘要
目的:在大肠杆菌中异源表达红霉素链霉菌聚酮合成酶eryAIII基因。方法:构建表达载体pET-m28a,PCR扩增高GC含量长片段基因eryAIII及分子伴侣GroELS的基因,并插入该载体,每个基因都能够独立启动和终止表达;将重组质粒转化至大肠杆菌BL21(DE3),用IPTG进行诱导表达。结果:NdeⅠ、HindⅢ分别酶切质粒pET-m28a/eryAIII-GroELS,琼脂糖凝胶电泳显示获得与预期大小相同的DNA片段;SDS-PAGE结果表明,重组大肠杆菌表达了由eryAIII编码的相对分子质量为348×103的蛋白;与GroELS共表达后,目的蛋白在上清中的表达量明显增加。结论:GroELS提高了eryAIII编码蛋白的可溶性,为红霉素合成通路在大肠杆菌中的重建奠定了基础。
Objective: To express the polyketide synthases gene eryAIII of Saccharopolyspora erythraea in Es-cherichia coli.Methods: Plasmid pET-28a was modified to construct vector pET-m28a used in this study.GC-rich eryAIII gene and molecular chaperon GroELS gene were amplified by PCR respectively,and the two expres-sion cassettes were inserted into the vector pET-m28a.The expression plasmid was transformed into E.coli BL21(DE3),and the IPTG was used to induce the expression of the recombinant proteins.Results: The result of agarose gel electrophoresis showed the expected size of DNA fragments produced by NdeⅠ or HindⅢ digestion of the constructed plasmid,indicating that the pET-m28a / eryAIII-GroELS was constructed successfully.The SDS-PAGE showed an expression protein band about 348 kD,suggesting that eryAIII gene was expressed after induction.The expression of protein in the supernatant increased significantly when it was coexpressed with GroELS.Conclusion: Solubility of protein coded by eryAIII was increased by coexpression of GroELS,which will greatly facilitate the ultimate reconstruction of erythromycin pathway in Escherichia coli.
出处
《生物技术通讯》
CAS
2010年第6期794-797,共4页
Letters in Biotechnology
基金
国家重大新药创制项目(2008ZXJ09007-001)