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

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。

Objective To carry out the secretive expression of natural N - terminal chicken cystatin （cC）. Methods Recombinant expression plasmid pPICZoA- cC was constructed by designing of distinct primers and inserting of cC cDNA into yeast expression vector pPICZoA. The recombinant cC was expressed in Pichia pastoris X - 33 after methanol induction. Results SDS - PAGE, Substrate SDS - PAGE and Native - PAGE analysis indicated that after the treatment of β - ME and GuHC1 with different combination, the destructive level of disulfide bond in recombinant cC is increased. Treatment by 5mol/ L GuHC1 and 10 % β - ME can break the intra - molecular disulfide bond almost entirely. Conclusion 92.9 % of the recom- binant cC had intra- molecular disulfide bonds and a similar inhibitory activity against DaDain with native cC.