关键词: 变异链球菌, 核糖-5-磷酸异构酶A, 融合蛋白, 原核表达, 蛋白纯化

Abstract: Objective To express, purify and characterize the ribose 5-phosphate isomerase A(rpiA) from Streptococcus mutans. Methods A DNA fragment encoding S. mutans ribose 5-phosphate isomerase A was amplified by PCR using the genomic DNA of Streptococcus mutans UA159 as a template. The PCR product was cloned into vector pGEX-6p-1. The construct carrying the coding DNA sequence of rpiA fused with GST was transformed into E.coli BL21(DE3), and the fusion protein was expressed by induction with IPTG. The recombinant protein was purified by affinity chromatography and ion exchange chromatography. The purified target protein was identified by SDS-PAGE and MALDI-TOF MS. Results S. mutans ribose 5-phosphate isomerase A was successfully expressed in E.coli in soluble form. After a series of purifications, we got the recombinant protein with the purity higher than 95%. The product was identified to be S. mutans ribose 5-phosphate isomerase A by SDS-PAGE and MALDI-TOF-TOF MS. Conclusion S. mutans ribose 5-phosphate isomerase A was successfully expressed in E.coli. An effective purification protocol was established. Recombinant rpiA with a purity higher than 95% was obtained, which provided a basis for the further studies of the biological activities and functions of ribose 5-phosphate isomerase A.

Key words: streptococcus mutans, ribose 5-phosphate isomerase A, fusion protein, prokaryotic expression, protein purification