Site-specific biotinylation of EGFP with double biotin molecules based on Avi-tag technology
10.3969/j.issn.1000-484X.2015.05.016
- VernacularTitle:基于 Avi-tag技术的双生物素分子位点专一性标记的增强型绿色荧光蛋白
- Author:
Shuangjia ZHAO
;
Rumeng BAO
;
Haike TANG
;
Xuecui BAO
;
Hongming YANG
;
Jinbao TANG
- Publication Type:Journal Article
- Keywords:
Enhanced green fluorescent protein;
Biotinylation;
Avi-tag;
BirA enzyme
- From:
Chinese Journal of Immunology
2015;(5):655-658
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To prepare the site-specific biotinylation of enhanced green fluorescence protein with double biotin molecules using Avi-tag technology.Methods:The EGFP gene was prepared by PCR and cloned into pdi-Avitag resulting the vector pEGFP-( Avitag) 2.The fusion protein EGFP-( Avitag ) 2 was expressed in E.coli DH5αand purified by employing IMAC.The site-specific biotinylation was implemented by BirA enzyme in vitro, and then was identified by competitive ELISA and Western blot.Results:The recombinant prokaryotic expression vector pEGFP-(Avitag)2 was correctly constructed,and EGFP-(Avitag)2 fusion was successfully expressed in E.coli DH5α.The results of competitive ELISA and Western blot showed that the EGFP-( Avitag) 2 could be site-specific biotinylation with double biotin molecules based on Avi-tag technology.Conclusion: The site-specific biotinylation of EGFP with double biotin molecules is successfully prepared,and we anticipate that can be used for BAS to improve the sensitivity and specificity of immunosensors.
