用P. pastoris的pAOX1表达系统表达木糖异构酶

Using pAOX1 Expression System to Express of Xylose Isomerase in P. pastoris

目的:应用P. pastoris的pAOX1表达系统分泌表达重组木糖异构酶。方法:用PCR法从大肠杆菌基因组中扩增木糖异构酶基因(xi)。用EcoRⅠ和NotⅠ双酶切将其基因克隆进P. pastoris表达载体。通过电转法将其木糖异构酶基因重组于P. pastoris基因组,筛选G418抗性700μg/ml的重组子作为工程菌GS115(pPIC9K-xi)。在摇瓶中发酵用甲醇诱导表达重组木糖异构酶。用SDS-PAGE分析重组蛋白的表达情况,用糖酵解法对表达产物进行活性分析。结果:木糖异构酶基因在pAOX1的调控下,在P. pastoris中经甲醇诱导能分泌表达,摇瓶发酵2d表达量为35mg/L,表达产物具有代谢木糖的作用。结论:成功地克隆了大肠杆菌的木糖异构酶基因,并实现用pAOX1系统在P. pastoris中表达中木糖异构酶,为用P. pastoris规模化生产重组木糖异构酶奠定了基础。

Objective：Using pAOX1 expression system of P.pastoris to express of xylose isomerase.Method：xylose isomerase（xi） gene was amplified from E.coli genome by PCR technique and cloned directly into the P.pastoris vector pPIC9K resulted in the expression plasmid of pPIC9K-xi which was then transformed into P.pastoris GS115 by electroporation method.A recombinant was selected as engineering strain GS115（pPIC9K-xi） from the YPD plate containing G418 700μg/ml to express xylose isomerase in sharking flash.SDS-PAGE was used to analyze the expressed products and the bioactivity was tested by glycolysis.Result：The target protein,Xylose Isomerase,expressed from the strain under the induction of methanol.The expression level was 35mg/L in 2 d fermentation in shaking flask,which showed the biological activity on metabolizing xylose.Conclusion：The gene of xi was successfully cloned from E.coli and achieved expression of recombinant XI protein in P.pastoris with the pAOX1 system,which has grounded on using P.pastoris to produce recombinant XI in large-scale.