Abstract: To investigate the function of the GhCOMT3 gene,the GhCOMT3 gene was insert into the pET-28a vector to construct fusion vector pET-28a-GhCOMT3,and transformed into E. coli BL21 (DE3) cells. The fusion protein could be induced to express by 0. 2 mmol /L IPTG,in the form of inclusion body at 16℃ for 4 h,30℃ for 3 h or 37℃ for 3 h. The most high expression quantity was induced at 30℃ for 4 h. Then to dissolved inclusion body,the supernatants were analyzed by SDS-PAGE and the results were identified with expecting. Then the protein was purified using His Trap^TMHP to get degenerated protein,SDS-PAGE and Western blotting were then employed to identify target protein,the results were identified with expecting. The prokaryotic expression system of pET-28a- COMT3 is successfully constructed in this experiment. The fusion protein was positive by Western blotting. So we can say that pET-28a-GhCOMT3 protein was expressed in E. coli. It can be used for further investigation on the function of the GhCOMT3 gene in protein level.

Key words: GhCOMT3, Prokaryotic expression, Protein purification, Western blotting

CLC Number: 

• Q78