红霉素链霉菌聚酮合成酶基因eryAIII的克隆及其在大肠杆菌中的表达

Cloning and Expression of Polyketide Synthases Gene eryAIII of Saccharopolyspora erythraea in Escherichia coli

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.