Site-specific biotinylation of EGFP with double biotin molecules based on Avi-tag technology / 中国免疫学杂志
Chinese Journal of Immunology
;
(12): 655-658, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-463440
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Immunology
Year:
2015
Type:
Article
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