發(fā)布時(shí)間：2018-05-01 02:11

  本文選題：精氨酸脫亞胺酶(ADI) + 隨機(jī)突變��； 參考：《江南大學(xué)》2013年碩士論文

【摘要】：精氨酸脫亞胺酶（arginine deiminase，ADI，EC3.5.3.6）是精氨酸營(yíng)養(yǎng)缺陷型腫瘤的熱門靶向治療藥物，并且因其對(duì)腫瘤細(xì)胞的高特異性以及對(duì)患者機(jī)體的低毒副作用而受到廣泛研究。然而當(dāng)前報(bào)道的ADI普遍在體內(nèi)生理?xiàng)l件下的應(yīng)用中存在諸多局限性，如酶活力低、底物親和性弱、半衰期短等，亟需采用蛋白質(zhì)工程進(jìn)行改造，使其成為在人體生理環(huán)境下酶學(xué)性能突出的藥用酶。 本研究室前期將自主篩選的一株ADI產(chǎn)生菌株P(guān)seudomonas plecoglossicida的ADI編碼基因在大腸桿菌中克隆表達(dá)后，經(jīng)一輪定向進(jìn)化后獲得一株酶學(xué)性能改良的ADI突變株M314(A128T/H404R/I410L)，，其在生理?xiàng)l件（pH7.35~7.45）下的酶活力較改造前（WT）提高近20倍，最適pH提高至6.5。隨后在突變熱點(diǎn)的研究中，獲得pH7.4下酶活力較M314提高了58%的優(yōu)勢(shì)突變株M13(D38H/A128T/E296K/H404R/I410L)。 為進(jìn)一步獲得生理?xiàng)l件下更具高應(yīng)用價(jià)值的ADI，本研究首先采用易錯(cuò)PCR建立隨機(jī)突變庫，通過高通量篩選得到突變株M173(A128T/H404R/I410L/K272R)，其在pH7.4下的比活較出發(fā)菌株M314提升了31.54%，且kcat/Km值提高了52.36%。同時(shí)采用半理性設(shè)計(jì)的方法，通過與底物L(fēng)-精氨酸結(jié)合前后的ADI三維結(jié)構(gòu)模型與ADI催化機(jī)制的解析，最終確定M01-Q162Y、M02-D166E、M03-L223I、M04-S245D、M05-R400M等五個(gè)位點(diǎn)氨基酸置換組合，并分別引入至M314進(jìn)行定點(diǎn)突變驗(yàn)證。于大腸桿菌中誘導(dǎo)表達(dá)后發(fā)現(xiàn)，M01-Q162Y、M02-D166E、M05-R400M三者的引入使酶活力大幅度下降，甚至完全喪失；然而， M04-S245D的疊加使得突變株M04(A128T/H404R/I410L/S245D) pH7.4下的比活在M314的基礎(chǔ)上提升了62.18%，kcat/Km值較其提高了37.12%；更重要的是，其最適pH由6.5升高至7.0，不僅更接近生理pH，且pH7.4下保留66%的酶活力；隨后，分別將通過隨機(jī)突變及定點(diǎn)突變所確定的優(yōu)勢(shì)氨基酸置換組合K272R與S245D疊加至突變株M13中，最終發(fā)現(xiàn)突變株M13-2(D38H/A128T/E296K/H404R/I410L/S245D)生理pH下的比活較M314提高了1.38倍，較M13提高了24.5%，最適pH也提高到7.0。最后，在突變株M13上建立關(guān)鍵位點(diǎn)162與245的飽和突變庫并進(jìn)行篩選，所得突變株M13+Q162S與M13+Q162T帶來了24%左右的可溶性蛋白量的增加，進(jìn)而使表觀酶活力提高了27%。針對(duì)以上的關(guān)鍵結(jié)果也進(jìn)行了相關(guān)機(jī)制分析。 本研究采用多種蛋白質(zhì)工程的方法成功地對(duì)ADI在生理pH條件下的性質(zhì)進(jìn)行了改良，不僅為蛋白質(zhì)的分子改造策略以及ADI蛋白的催化機(jī)制與可溶性表達(dá)的研究提供實(shí)驗(yàn)思路，也對(duì)開發(fā)有良好應(yīng)用前景和自主知識(shí)產(chǎn)權(quán)的蛋白質(zhì)抗癌新藥有重要的意義。
[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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：Q55

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本文編號(hào)：1827256