重组猪胰蛋白酶及其R122位点突变体在毕赤酵母中的高效表达及其性质研究

High-level Expression and Characterization of Recombinant Porcine Trypsin and Its R122 Site Mutant in Pichia pastoris

目的：研究R122位点突变重组猪胰蛋白酶,与野生型酶相比较,该位点对重组猪胰蛋白酶（RPT）性质的影响。方法：以毕赤酵母GS115作为表达宿主,对RPT、突变体mRPT（R122H）和mRPT（R122H/R73G/R130T）进行表达及纯化。并对其性质和稳定进行对比研究。结果：重组胰蛋白酶及其突变体在毕赤酵母中均获得了高效表达。相对于RPT,突变体mRPT（R122H）和mRPT（R122H/R73G/R130T）在以N-苯甲酰-L-精氨酸乙酯（BAEE）为底物时,具有更强底物结合力,三者的米氏常数分别为18.8μmol/L、9.0μmol/L和11.0μmol/L;两突变体耐高温耐碱能力增强;在Ca2＋存在及去除的条件下,突变体具有更强的抗自降解能力。结论：可以利用毕赤酵母高效表达重组胰蛋白酶及其突变体。mRPT（R122H）和mRPT（R122H/R73G/R130T）相对于野生型RPT,对高p H条件和高温的耐受性增强,该稳定性的提高主要归因于R122位点的突变。

Objective： Study the effect of R122 residue mutation on the stability of recombinant porcine trypsin（ RPT）. Methods： RPT,mutants mRPT（ R122 H） and mRPT（ R122 H/R73 G/R130） were expressed in Pichia pastoris GS115 and further purified. The properties and stabilities of RPT and two mutants was investigated and compared. Results： RPT and its mutants were highly expressed in Pichia pastoris. Relative to RPT,mutant mRPT（ R122 H） and mRPT（ R122 H/R73 G/R130 T） showed higher affinity to substrate BAEE,The Kmvalues were 18. 8μmol/L, 9. 0μmol/L and 11. 0μmol/L, respectively. Increased stability of mutants to high temperature and alkali were observed. And higher resistance against autolysis were got in the presence and without Ca2 ＋. Conclusion： Pichia pastoris can be used to efficiently express RPT and its mutants. Increased stability under alkaline condition and higher thermal stability and higher anti-self-digestion were got in mutant mRPT（ R122 H） and mRPT（ R122 H/R73 G/R130 T） compared to wild-type RPT,which contribute to the site mutation at the R122.