Determination of binding ability of PaP3’s terminase large subunit to cos site
- VernacularTitle:铜绿假单胞菌噬菌体PaP3末端酶大亚单位DNA结合能力的测定
- Author:
Xiaodong SHEN
;
Kebin ZHANG
;
Ming LI
;
Yingbing ZHOU
;
Rui JIAN
;
Xiaomei HU
;
Zhijin CHEN
;
Xiancai RAO
;
Fuqua HU
- Publication Type:Journal Article
- Keywords:
terminase large subunit;
bacteriophage PaP3;
EMSA;
protein expression
- From:Journal of Third Military Medical University
2003;0(22):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the binding ability of the terminase large subunit of Pseudomonas aeruginosa bacteriophage PaP3 to the cos site. Methods The gene tls was amplified from the genome of bacteriophage PaP3 by PCR and subcloned into pMD18-T vector. Then the gene tls cut down from the vector was inserted into the plasmid pQE31 which could give a 6-His tag at the N-terminal of the expressed protein. The recombinant vector pQE-tls was transformed to E.coli. JM109, after induction with IPTG, the expressed bacteria were resuspended and sonicated, then after centrifugation, the inclusion body was obtained. The inclusion body was dissolved with lysis buffer, then the tagged protein was purified by Ni-NTA affinity chromatography and renatured by dialysis. Finally the DNA-binding ability of the fusion protein rTLS was determined by EMSA. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. After purification and renaturation, the fusion protein rTLS can partially bind the cos fragment. Conclusion The fusion protein rTLS was successfully expressed, purified and renatured. The rTLS has the specific DNA-binding activity. The present work lays the foundation for the further research of the gene tls.
