科研之友
微信
新浪微博
Facebook
分享链接
摘要
Pichia pastoris is widely used as a platform for heterologous protein expression because of its high volumetric productivity. Multicopy integration of the target gene is commonly used to improve the production of the target protein. Cre/lox recombination system is a powerful tool for the marker rescue during multiple integrations with one selection marker. Here we reported a novel expression vector based on the Cre/lox recombination system for multiple integrations of target gene to construct multicopy expression strain of P. pastoris. P-AOX1 promoter was fused to cre to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in Escherichia coli was blocked by introducing the operator gene lacO. The expression vector designed pMCO-AOX alpha was stable in E. coli and could effectively rescue the Zeocin resistance gene for next round of integration in P. pastoris. Phytase AppA from E. coli was chosen as a reporter gene. Transformants with 2-16 copies of appA were constructed by using a single antibiotic. Expression of appA was gene dosage dependent when <12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of appA were integrated comparing with the single copy integration. Our results showed that pMCO-AOX alpha was highly effective for rational construction of multicopy transformat in P. pastoris.
- 出版日期2017-9-11
- 单位南京农业大学
