High-level expression and purification of ComE in Escherichia coli
- VernacularTitle:变形链球菌ComE蛋白在大肠杆菌中的高效表达与纯化
- Author:
Lei PENG
;
Xiaodi LIU
;
Lihong GUO
- Publication Type:Journal Article
- Keywords:
Streptococcus mutans;
Prokaryotic expression;
Protein purification;
Quorum sensing
- From:
Journal of Practical Stomatology
1996;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the prokaryotic expression vector pET28a(+)-comE, to get the highest expression of comE in E. coli BL21(DE3), and to obtain the purified ComE fusion protein. Methods: ComE gene was amplified by PCR with specific primers from genome of Streptococcus mutans UA159. After digested by two restriction endonucleases BamH Ⅰand Xho Ⅰ, the gene segment was inserted into vector pET28a(+). Then the recombinant vector was transformed into E. coli BL21(DE3). The protein expression was induced by IPTG and the result was confirmed by Western blot. The solubility of the fusion protein was examined by 12% SDS-PAGE, and the expression conditions were optimized. Finally the fusion protein was purified by a two-step purification procedure utilizing Ni2+ chelating column and S75 size exclusion column. Results: The recombinant plasmid was confirmed by PCR, restriction endonucleases digestion and sequence analysis, which showed that the inserted segment was identical to comE gene reported by GenBank without nucleotide mutation. The fusion protein which was confirmed by Western blot can be expressed in E. coli BL21(DE3) and existed both in supernatant and precipitation. The maximum expression product amount was obtained when the A600 value of E. coli BL21(DE3) was 0.4, IPTG concentration was 0.10 mmol/L and induction time was 4 h. Conclusion: The recombinant plasmid is constructed and the ComE fusion protein is purified by the optimum conditions.
