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| A Secretive Pichia pastoris Expression Vector Capable of Direct PCR Product Cloning |
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Abstract It is difficult to obtain N-terminus-authentic recombinant protein in secretive expression system, for cloning of the objective gene through restriction and ligation inevitably introduces additional amino acids between the objective protein and the secretive signal peptide. This work proposed a novel approach to address this problem in the Pichia pastoris secretive expression system through construction and application of an expressive T-vector. A randomly selected fragment was PCR amplified with properly designed primers, such that Xho⒐and Eam1105⒐ restriction sites were included in the 5ˇ end of the amplified product, and Eam1105⒐ and Xba⒐ restriction sites were included in the 3ˇ end. The PCR amplified product was inserted into the P. pastoris expression plasmid pPICZ冄A through Xho⒐ and Xba⒐ restriction sites. The resultant plasmid was digested with Eam1105⒐, and the big fragment was recovered, generating the P. pastoris expressive T-vector pPICZT. The gene of cellobiohydrolase ⒑of T. reesei was expressed in P. pastoris with this expressive T-vector. The results indicated that the constructed expression T-vector was convenient for PCR product cloning, and was effective for heterolgous protein expression in P. pastoris. On the other hand, application of the expression T-vector avoided the introduction of additional amino acids in the N-terminus of the expressed protein, an event that generally occurred when normal expression vectors were used in secretive expression system.
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Received: 17 August 2006
Published: 10 May 2010
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