【作者】 杨帆；

【导师】 李素霞；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学（专业学位）， 2021， 硕士

【摘要】 Kex2蛋白酶(Kexin EC.3.4.21.61)是一种钙离子依赖的丝氨酸蛋白水解酶,特异性识别并切割两个连续碱性氨基酸(-ArgArg-或-LysArg-)的羧基端肽键。本论文为了解决天然Kex2蛋白酶的不稳定问题,首先研究了 N端前肽和C末端结构域对Kex2蛋白酶活性和稳定性的影响。结果发现去除前肽结构域的Kex2蛋白酶无生物活性,证明了前肽对于Kex2蛋白酶活性的重要性。C末端结构域的增加不直接影响Kex2蛋白酶的活性,但会影响Kex2蛋白酶在酵母中的分泌表达。最终确定含有前肽并去除C末端结构域的序列长度为667个氨基酸的Kex2蛋白酶作为基础蛋白,进一步进行稳定性研究。三维结构分析表明:R207、R542和K291这三个位点均有可能是Kex2蛋白酶潜在的自降解位点,因此构建了 Kex2蛋白酶的三种突变体:R207系列突变体、R542系列突变体以及K291系列突变体。Kex2蛋白酶的突变体在毕赤酵母GS115中全部以外分泌的形式表达,利用Q-FF离子交换层析纯化获得目的蛋白。结果表明,Kex2蛋白酶的R207系列和R542系列突变体仍存在类似的降解问题,Kex2蛋白酶的K291系列突变体的稳定性提高,并在很大程度上解决了自降解的问题。对比研究了 Kex2-K291的四种突变体Kex2-K291H、Kex2-K291L、Kex2-K291I和 Kex2-K291Q,其中 Kex2-K291H 突变体的稳定性最好,因此对Kex2-K291H突变体进行了详细的研究,为该酶的实际应用提供一些参考。更多还原

【Abstract】 Kex2 protease(Kexin EC.3.4.21.61)is a calcium ion-dependent serine proteolytic enzyme that specifically recognizes and cleaves at the carboxy terminal peptide bond of two consecutive basic amino acids(such as-ArgArg-or-LysArg-).In order to solve the instability problem of natural Kex2 protease,this paper first studied the influence of N-terminal propeptide and C-terminal domain on the activity and stability of Kex2 protease.As a result,it was found that the Kex2 protease without the propeptide domain had no biological activity,which proved the importance of the propeptide to the activity of Kex2 protease.The increase of the C-terminal domain didn’t directly affect the activity of Kex2 protease,but it would affect the secretion and expression of Kex2 protease in yeast.Finally,the Kex2 protease with a sequence length of 667 amino acids containing the propeptide and removing the C-terminal domain was determined as the basic protein,and further stability studies were carried out.The three-dimensional structure analysis showed that the three sites of R207,R542 and K291 might be potential autodegradation sites of Kex2 protease.Therefore,three kinds of mutants of Kex2 protease were constructed:R207 series mutants,R542 series mutants and K291 Series of mutants.The mutant of Kex2 protease was expressed in the form of all but secretion in Pichia pas toris GS115,and these proteases were obtained by Q-FF ion exchange chromatography.Compared with the original sequence,the R207 series and the R542 series mutants of Kex2 protease still had similar degradation problems.It was inferred that the R207 and R542 sites were not potential autodegradation sites of Kex2 protease;the stability of the K291 series mutants of Kex2 protease were improved and the problem of self-degradation was solved to a large extent.Among the four constructed mutants Kex2-K291H,Kex2-K291L,Kex2-K291I and Kex2-K291Q,the Kex2-K291H mutant had the best stability.Therefore,a more detailed application study was carried out on the Kex2-K291H mutant.It may provide some references for the practical application of the enzyme.更多还原