随机突变结合半理性设计改造精氨酸脱亚胺酶

发布时间：2018-05-01 02:11

本文选题：精氨酸脱亚胺酶(ADI) + 随机突变　；参考：《江南大学》2013年硕士论文

【摘要】：精氨酸脱亚胺酶（arginine deiminase，ADI，EC3.5.3.6）是精氨酸营养缺陷型肿瘤的热门靶向治疗药物，并且因其对肿瘤细胞的高特异性以及对患者机体的低毒副作用而受到广泛研究。然而当前报道的ADI普遍在体内生理条件下的应用中存在诸多局限性，如酶活力低、底物亲和性弱、半衰期短等，亟需采用蛋白质工程进行改造，使其成为在人体生理环境下酶学性能突出的药用酶。本研究室前期将自主筛选的一株ADI产生菌株Pseudomonas plecoglossicida的ADI编码基因在大肠杆菌中克隆表达后，经一轮定向进化后获得一株酶学性能改良的ADI突变株M314(A128T/H404R/I410L)，，其在生理条件（pH7.35~7.45）下的酶活力较改造前（WT）提高近20倍，最适pH提高至6.5。随后在突变热点的研究中，获得pH7.4下酶活力较M314提高了58%的优势突变株M13(D38H/A128T/E296K/H404R/I410L)。为进一步获得生理条件下更具高应用价值的ADI，本研究首先采用易错PCR建立随机突变库，通过高通量筛选得到突变株M173(A128T/H404R/I410L/K272R)，其在pH7.4下的比活较出发菌株M314提升了31.54%，且kcat/Km值提高了52.36%。同时采用半理性设计的方法，通过与底物L-精氨酸结合前后的ADI三维结构模型与ADI催化机制的解析，最终确定M01-Q162Y、M02-D166E、M03-L223I、M04-S245D、M05-R400M等五个位点氨基酸置换组合，并分别引入至M314进行定点突变验证。于大肠杆菌中诱导表达后发现，M01-Q162Y、M02-D166E、M05-R400M三者的引入使酶活力大幅度下降，甚至完全丧失；然而， M04-S245D的叠加使得突变株M04(A128T/H404R/I410L/S245D) pH7.4下的比活在M314的基础上提升了62.18%，kcat/Km值较其提高了37.12%；更重要的是，其最适pH由6.5升高至7.0，不仅更接近生理pH，且pH7.4下保留66%的酶活力；随后，分别将通过随机突变及定点突变所确定的优势氨基酸置换组合K272R与S245D叠加至突变株M13中，最终发现突变株M13-2(D38H/A128T/E296K/H404R/I410L/S245D)生理pH下的比活较M314提高了1.38倍，较M13提高了24.5%，最适pH也提高到7.0。最后，在突变株M13上建立关键位点162与245的饱和突变库并进行筛选，所得突变株M13+Q162S与M13+Q162T带来了24%左右的可溶性蛋白量的增加，进而使表观酶活力提高了27%。针对以上的关键结果也进行了相关机制分析。本研究采用多种蛋白质工程的方法成功地对ADI在生理pH条件下的性质进行了改良，不仅为蛋白质的分子改造策略以及ADI蛋白的催化机制与可溶性表达的研究提供实验思路，也对开发有良好应用前景和自主知识产权的蛋白质抗癌新药有重要的意义。
[Abstract]:Arginine deiminase EC3.5.3.6) is a popular target drug for arginine nutrition-deficient tumors, and has been widely studied for its high specificity to tumor cells and low toxicity and side effects to patients. However, there are many limitations in the application of ADI in vivo physiological conditions, such as low enzyme activity, weak substrate affinity, short half-life and so on. It has become a medicinal enzyme with outstanding enzymatic properties in human physiological environment. In our lab, we cloned and expressed the ADI encoding gene of Pseudomonas plecoglossicida, a ADI producing strain, in Escherichia coli. A modified ADI mutant M314A128T / H404R / I410L was obtained after a round of directed evolution. The enzyme activity of M314A128T / H404R / I410LN under physiological conditions (pH 7.35 ~ 7.45) was nearly 20 times higher than that before modification, and the optimum pH was increased to 6.5. Then in the study of mutation hotspots, the dominant mutant strain M13D38H / A128T / E296K / H404R / I410L was obtained, which showed that the enzyme activity under pH7.4 was 58% higher than that of M314, and the dominant mutant strain M13D38H / A128T / E296K / H404R / I410L was obtained. In order to further obtain the more practical value of ADI under physiological conditions, the random mutation library was established by error-prone PCR, and the mutant strain M173A128T / H404R / I410L / K272RN was obtained by high-throughput screening. The specific activity of the mutant under pH7.4 was 31.54 higher than that of the original strain M314, and the kcat/Km value was increased by 52.36B. At the same time, by using the semi-rational design method, the ADI three-dimensional structure model before and after binding with the substrate L-arginine and the analysis of the catalytic mechanism of ADI were used to determine the amino acid substitution combinations of M01-Q162YTX M02-D166EN M03-L223IHM04-S245DKM05-R400M. And introduced to M314 to verify the site-directed mutation. After induced expression in Escherichia coli, it was found that the introduction of M01-Q162YN M02-D166EN M05-R400M significantly decreased or even completely lost the enzyme activity; however, the superposition of M04-S245D increased the specific activity of the mutant M04A128T / H404RI410L / S245D pH7.4 by 62.18kcat / km higher than M314; more importantly, M04A128T / H404RI410L / S245D) pH7.4 increased the specific activity of M01-Q162YN M02-D166EN M05-R400M; more importantly, The optimal pH was increased from 6.5 to 7.0, which was not only closer to physiological pH, but also reserved 66% of enzyme activity under pH7.4. Subsequently, K272R, a dominant amino acid replacement combination determined by random mutation and site-directed mutation, and S245D were superimposed on the mutant strain M13, respectively. It was found that the specific activity of M13-2D38H / A128T / E296K / H404R / I410L / S245D at physiological pH was 1.38 times higher than that of M314, 24.5g higher than M13, and the optimum pH was 7.0. Finally, the saturated mutants of 162 and 245 were established and screened on the mutant M13. The mutants M13Q162S and M13Q162T increased the amount of soluble protein by about 24%, and the apparent enzyme activity increased by 27%. In view of the above key results, the related mechanism is also analyzed. In this study, the properties of ADI under physiological pH conditions were successfully improved by various protein engineering methods, which not only provided experimental ideas for the study of protein molecular transformation strategy and the catalytic mechanism and soluble expression of ADI protein. It is also of great significance for the development of new anti-cancer protein drugs with good application prospects and independent intellectual property rights.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q55

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