天然氮末端重组cC可溶性分泌表达
Soluble expression and secretion of natural N-terminal recombinant chicken cystatin
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摘要: 目的 构建具有天然氮末端的重组鸡半胱氨酸蛋白酶抑制剂(chicken cystatin,cC)在毕赤酵母中高效分泌表达。方法 通过设计独特的引物,利用DNA重组技术将cC的cDNA片段插入分泌型酵母表达载体pPICZαA中,构建相应的重组酵母表达质粒pPICZαA-cC,并在巴氏毕赤酵母(Pichia pastoris)菌株X-33中甲醇诱导表达。结果 经十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、底物活性SDS-PAGE和Native-PAGE分析,随着不同剂量的β-巯基乙醇和盐酸胍组合处理重组cC,二硫键被破坏的程度加深,5 mol/L盐酸胍与10%β-巯基乙醇的组合几乎完全打开了分子内的二硫键。结论 92.9%的重组cC具有二硫键并与天然cC具有相似的木瓜蛋白酶抑制活性。Abstract: Objective To carry out the secretive expression of natural N-terminal chicken cystatin(cC).Methods Recombinant expression plasmid p PICZαA-cC was constructed by designing of distinct primers and inserting of cC cDNA into yeast expression vector p PICZαA.The recombinant cC was expressed in Pichia pastoris X-33 after methanol induction.Results SDS-PAGE,Substrate SDS-PAGE and Native-PA GE analysis indicated that after the treatment of β-ME and GuHCl with different combination,the destructive level of disulfide bond in recombinant cC is increased.Treatment by 5mol/L GuHCl and 10%β-ME can break the intra-molecular disulfide bond almost entirely.Conclusion 92.9% of the recombinant cC had intra-molecular disulfide bonds and a similar inhibitory activity against papain with native cC.