本文選題：精氨酸脫亞胺酶 + 定點(diǎn)突變��； 參考：《江南大學(xué)》2012年碩士論文

【摘要】：精氨酸脫亞胺酶(ADI)是一種針對(duì)精氨酸缺陷型癌癥(如:肝癌、黑素瘤)的新藥,目前處于臨床三期試驗(yàn)。為了進(jìn)一步探明M314中關(guān)鍵位點(diǎn)對(duì)精氨酸脫亞胺酶的作用,本研究通過定點(diǎn)突變技術(shù)分析了精氨酸脫亞胺酶的特定氨基酸位點(diǎn)對(duì)酶活力的影響機(jī)制。 針對(duì)已報(bào)道的關(guān)鍵氨基酸殘基A128,H404,I410,采用QuikChange法進(jìn)行定點(diǎn)突變,獲得ADI突變株M1 (A128T)、M2 (H404R)、M3 (I410L)和M4 (A128T/H404R)。將突變株在大腸桿菌BL21 (DE3)中進(jìn)行重組表達(dá),并對(duì)純化獲得的突變蛋白進(jìn)行酶學(xué)性質(zhì)研究。結(jié)果表明,突變位點(diǎn)A128T和H404R對(duì)ADI最適pH的提高,生理中性(pH 7.4)條件下的酶活力和穩(wěn)定性的提高,以及Km值的降低均具有顯著的作用。 為了進(jìn)一步改善ADI的酶學(xué)性質(zhì),結(jié)合序列分析以及相關(guān)文獻(xiàn)報(bào)道,選定V10F、R18L、D38H、D44E、A128R、E296K作為突變位點(diǎn),以WT-ADI為出發(fā)菌株進(jìn)行單點(diǎn)突變,獲得突變株后并對(duì)各個(gè)突變酶的酶學(xué)性質(zhì)進(jìn)行了考察,結(jié)果表明,D38H和E296K對(duì)WT-ADI的酶活力的提高最具有顯著的促進(jìn)作用。將突變熱點(diǎn)D38H和E296K引入到突變株M314中,獲得了突變株M11 (D38H/A128T/H404R/I410L)、M12 (A128T/E296K/H404R/I410L)和M13 (D38H/A128T/E296K/H404R/I410L),其中M13 (D38H/A128T/E296K/H404R/I410L)在生理中性條件下(pH 7.4)酶活力較M314(10.08 U/mg)提高了58%,比活力為17.02 U/mg,最適pH為6.5。M13的Km(0.57 mmol/L)較M314(0.67 mmol/L)有所降低,其kcat/Km較M314有顯著提高。結(jié)果表明,突變株M13與底物的親和力以及對(duì)底物的催化效率方面均有較大提高。 以來源于Pseudomonas aeruginosa的ADI蛋白質(zhì)結(jié)構(gòu)為模型,采用Swiss-Model對(duì)來源于P. plecoglossicida的ADI突變株M314進(jìn)行同源結(jié)構(gòu)建模,并對(duì)各突變位點(diǎn)V10F、R18L、D38H、D44E、A128R、A128T、E296K、H404R和I410L的立體結(jié)構(gòu)進(jìn)行分析。本研究為闡明ADI的酶活力影響機(jī)制和蛋白質(zhì)的理性改造提供了一定的依據(jù)。
[Abstract]:Arginine deiminase (ADI) is a new drug for arginine deficient cancer (such as liver cancer, melanoma). In order to further investigate the effect of key sites in M314 on arginine deiminase, In this study, the effects of specific amino acid sites of arginine deiminase on the activity of arginine deiminase were analyzed by site-directed mutagenesis. Based on the reported key amino acid residues A128T404H40410, the directed mutagenesis was carried out by QuikChange method. The mutant was expressed in Escherichia coli BL21 (DE3) and the purified mutant protein was studied by enzymatic properties. The results showed that the mutation sites A128T and H404R had significant effects on the increase of the optimum pH of ADI, the increase of enzyme activity and stability under physiological neutral pH 7.4) and the decrease of km value. In order to further improve the enzymatic properties of ADI, A128T and H404R had significant effects on ADI. Combined with sequence analysis and related literature reports, we selected V10FN R18LN D38HN D44EA128RN E296K as the mutation site and WT-ADI as the starting strain for single point mutation. After obtaining the mutant strain, the enzymatic properties of each mutant enzyme were investigated. The results showed that D38H and E296K enhanced the activity of WT-ADI most significantly. 灝嗙獊鍙樼儹鐐笵38H鍜孍296K寮曞叆鍒扮獊鍙樻牚M314涓，

本文編號(hào)：2006941