Expression and purification of four single-stranded DNA-binding proteins and their binding on HCV RNA
10.3760/cma.j.issn.1003-9279.2013.05.011
- VernacularTitle:四种SSB蛋白的表达纯化及其对HCV RNA的绑定分析
- Author:
Hai-Yan SHI
1
;
Yong-Jun LI
;
Ji-Min GAO
Author Information
1. 325035,温州医科大学检验医学院生命科学院
- Keywords:
DNA;
Expressed sequence tags;
Hepatitis C;
RNA
- From:
Chinese Journal of Experimental and Clinical Virology
2013;27(5):354-356
- CountryChina
- Language:Chinese
-
Abstract:
Objective Express and purify four single-stranded DNA-binding (SSB) proteins,and evaluate the binding of SSB proteins on HCV RNA.Methods The expression plasmids of four SSB proteins were conducted,termed TTH,SSOB,KOD and BL21,respectively.The BL21 (DE3) was transformed by the expression plasmid of TTH,Transetta (DE3) were transformed by the expression plasmid of SSOB,KOD and BL21,then protein expression was induced with IPTG,the expression products were analysised by SDS-PAGE.To evaluate the binding of SSB on HCV RNA,RNA-SSB protein complexes were applied to a 1.2％ TAE agarose gel.Results Suitable competent cells were transformed with the expression plasmids,induced by IPTG.SSB proteins were purified by affinity chromatography,to visualize their purity all SSB proteins were applied to SDS-PAGE analysis.All four proteins showed single clear bands.We have successfully obtained the SSB protein expression plasmid,expressed and purified SSB protein.TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity.The each of SSB-RNA complex migrated more slowly than the sole RNA,which suggested SSB protein could specifically bind to RNA.Conclusions We have expressed and purified four SSB proteins,and for the first time found that SSB protein can bind HCV RNA.Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.
