不同分泌信号对豹蛙酶在巴斯德毕赤酵母中分泌效率的影响

Effects of Different Secretion Signals on Efficiency of Onconase Secretion in Pichia pastoris

目的:研究不同分泌信号对豹蛙酶(ONC)在巴斯德毕赤酵母中分泌效率的影响。方法:根据已知天然ONC的氨基酸序列,结合酵母偏爱密码子,设计并合成了ONC基因序列,通过融合PCR获得了不同分泌信号与ONC的融合基因,将其克隆至表达载体pPIC9k中,然后将线性化的重组表达载体电击转化GS115毕赤酵母感受态细胞,通过SDS-PAGE检测目的蛋白表达量。结果:糖基化ONC表观相对分子质量为(14～16)×103,且其糖基化不均匀,非糖基化蛋白肽链的相对分子质量约为12×103;采用不含EAEA结构的α交配因子作为分泌信号时ONC没有表达,利用含EAEA结构的α交配因子和α交配因子的pre肽作为分泌信号时均能够显著提高其表达量。结论:采用含EAEA结构的α交配因子和α交配因子的pre肽作为分泌信号均能够显著提高ONC在巴斯德毕赤酵母中的表达量。

Objective： To investigate the effects of different secretion signals on efficiency of onconase（ONC） secretion in Pichia pastoris expression system. Methods： The cDNA encoding ONC was synthesized with a yeast bias codon, ONC gene with different secretion signals were inserted into pPIC9k, then linearized plasmids were transformed into P.pastoris GS115 cells by electroporation. An engineering strain which could express ONC was finally screened from the transformants by SDS-PAGE. Results： SDS-PAGE analysis revealed that the glycosylated ONC was heterogeneous and migrating at the expected molecular weight range of 14-16 kD, and the non-glycosylated ONC was at about 12 kD. Both α mating factor （with EAEA space） and its pre peptide are much better than α mating factor without EAEA spacer as secretive signal for the expression of ONC in P.pastoris expression system. Conclusion： Both α mating factor（with EAEA space） and its pre peptide can lead high yield of ONC in P.pastoris.