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

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Ca ²⁺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pH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。

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

Objective:Study the effect of R122 residue mutation on the stability of recombinant porcine trypsin (RPT).Methods:RPT,mutants mRPT(R122H) and mRPT(R122H/R73G/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 (R122H) and mRPT (R122H/R73G/R130T) showed higher affinity to substrate BAEE, The K_m values 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 Ca ²⁺. 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 (R122H) and mRPT (R122H/R73G/R130T) compared to wild-type RPT, which contribute to the site mutation at the R122.