A Secretive Pichia pastoris Expression Vector for Direct PCR Product Cloning
- VernacularTitle:可直接克隆PCR产物的毕赤酵母分泌型表达载体
- Author:
Chao ZHANG
;
Gang LIU
;
Shao-Wen YU
;
Miao XING
;
- Publication Type:Journal Article
- Keywords:
Cellobiohydrolase Pichia pastoris Secretive expression vector Trichoderma reesei Tvector
- From:
China Biotechnology
2006;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
A secretive expression vector of Pichia pastoris system which can be used for the direct cloning of PCR products was constructed,and was verified through the expression of recombinant cellobiohydrolase II in Pichia pastoris.A randomly selected fragment was amplified with properly designed primers by PCR.The XhoI and Eam1105Ⅰ restriction sites were included in the 5'end of the fragment,and the Eam1105Ⅰ and XbaI restriction sites were included in its 3'end.The PCR amplified product was inserted into the P.pastoris expression plasmid pPICZ?A through XhoI and XbaI restriction sites and the resultant plasmid was digested with Eam1105Ⅰ,and lastly the big fragment was recovered,generating the P.pastoris expressive Tvector pPICZ?T.Then the cellobiohydrolase II of T.reesei was successfully expressed in P.pastoris with this expressive Tvector.Such results indicated that the constructed expression Tvector was convenient for PCR product cloning,and was effective for heterologous protein expression in P.pastoris.On the other hand,the application of the expression Tvector avoided the introduction of additional amino acids at the Nterminus of the expressed protein,which generally occurred when normal expression vectors were used in secretive expression system.
