Optimization The Expression Vector PPIC9k Of Pichia Pastoris

As a eukaryotic expression system, methanol nutrition type Pichia pastoris has been more and more used to express heterologous proteins. Compared with other expression systems, it is easy to operate, suitable for high density fermention. Moreover, the proteins expressed in P. pastoris are secreted and soluble, glycosylated or another post-translational modificated, which will help to form bioactive proteins. This study has constructed a series of expression vectors on the basis of the current commercializated expression vector pPIC9k.Considerating the safety of genetically engineered strains and cloning strategies, such as pHBM905A、pHBM905B、pHBM905D、pHBM905M, this study aims to construct some vectors from pPIC9k. Firstly, component of "Ampr+Ori" was inserted into the HIS4gene; and the resistance gene Kanr was inserted into the multiple cloning sites, recognition sequences of Not I and Cpo I enzymes were intered into3’and5’end of Kanr gene, respectively. This new vector has named pHBM905A. The target gene was cloned into the vector pHBM905A dependent on the T4DNA polymerase3’-5’exonuclease activity. Then, the AOX1promoter of pHBM905A was replaced by mutant d1+2×201AOXl promoter, and this modified vector named pHBM905D. Subsequently, the Saccaromyces cerevisiae MFa-SS signal sequences on pHBM905A was replaced by MF4I-SS signal sequences synthesized according to the codon preference of P. pastoris, and this modified vector named pHBM905B. Finally, MFa-SS signal sequences on pHBM905D were replaced by MF4I-SS signal sequences, and this modified vector named pHBM905M.Another aim of this study is to construct a new vector that can build expression cassette multiple copies in vitro, based on pHBM905M vector and Biobrick method. First of all, Xba I recognized sequences on the pHBM905M were mutated. Then, EcoR I+Xba I and Spe I+BamH I recognized sites would be inserted into the5’and3’end of the expression cassette, respectively. Thus, the vector pHBM905BDM can be obtained. The recombinant plasmid with expression cassette multiple copies could be obtained after repeated digestion and ligation. Subsequently, recombinant plasmids were linearized with Sal I and transformed into P. pastoris GS115. Now the recombinant strains with expression cassette multiple copies were obtained.These five vectors were all validated by cloning and expression. According to the results, d1+2×201AOX1promoter and MF4I-SS signal have a synergistic effect. In improving the heterologous protein expression, the combined role has the more effect than the single role. Moreover, the heterologous expression was higher and higher with the increased copy numbers within a certain range.