Determination of binding ability of PaP3’s terminase large subunit to cos site / 第三军医大学学报

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.

Experimental identification of endonuclease activity of the putative gene tls from phage PaP3 / 解放军医学杂志

Objective To experimentally verify the putative function of gene tls from Pseudomonas aeruginosa phage PaP3. Methods The gene tls was amplified from the genome of phage 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 the 6-His tag at the N' side of the expressed protein. The recombinant vector pQE-tls was transformed to E. coli JM109. After induction with IPTG, the expressed bacteria was resuspended and sonicated, then the inclusion body was obtained after centrifugalization. The inclusion body was then dissolved with lysis buffer. The inclusion body solution, which has the target protein, was then purified by affinity chromatography and renatured by dialysis. Finally the nuclease activity of the fusion protein H6-TLS was tested on the plasmid pMD-cos which contains the cutting site of terminase large subunit. Results The expression plasmid pQE31-tls was successfully constructed, and the target protein yield was up to 30% of the total bacterial proteins. Meanwhile the substrate vector pMD-cos was also successfully constructed. After purification and renaturation, the fusion protein H6-TLS could partially cut the substrate vector pMD-cos. Conclusion The fusion protein H6-TLS was successfully expressed, purified and renatured, and it was shown to possess nuclease function. The experiment lays the foundation for further research of the gene tls.