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首页> 外文期刊>Preparative biochemistry & biotechnology: An international journal for rapid communication >Co-overexpression of folding modulators miproves the solubility of the recombinant guinea pig liver transglutaminase expressed in escherichia coli
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Co-overexpression of folding modulators miproves the solubility of the recombinant guinea pig liver transglutaminase expressed in escherichia coli

机译：折叠调节剂的共过量表达提高了在大肠杆菌中表达的重组豚鼠肝脏转谷氨酰胺酶的溶解度

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摘要

Transglutaminases (EC 2.3.2.13) catalyze the formation of #epsilon#-(#gamma#-glutamyl)lysine cross-links and the substitution of primary amines for the #gamma#-carboxamide groups of protein bound glutamine.residues,and are involved in many biolgical phenomena.Transglutaminase reactions are also applicable in applied enzymoogy.Here,we estabilised an expression system of recombinant mammalian tissue-type transglutaminase with high productivity.Overexpression of guinea pig liver transglutaminase in Escherichia coli,using a plasmid pET21-d,mostly resulted in the accumulation of insoluble and inactive enzyme protein.By the expression culture at lower temperatures (25 and 18 deg C).however,a fractionof the soluble and actie enzyme protein slightly increased.Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E.coli cells.The specific activity,the affinity to the amine substrate,and the sensitiviy to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme.These results indicated that co-overexpression of folding modulators test improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase,but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.
机译：转谷氨酰胺酶（EC 2.3.2.13）催化#epsilon＃-（＃gamma＃-谷氨酰胺基）赖氨酸交联的形成以及伯胺取代蛋白结合的谷氨酰胺，残基的＃gamma-羧酰胺基，并且涉及转谷氨酰胺酶反应也适用于应用的酶法。在此，我们稳定了重组哺乳动物组织型转谷氨酰胺酶的表达系统，提高了生产率。豚鼠肝转谷氨酰胺酶在大肠杆菌中的过表达，主要使用质粒pET21-d导致不溶性和非活性酶蛋白的积累。通过在较低温度（25和18摄氏度）下进行表达培养，可溶和肌动酶蛋白的比例略有增加。分子伴侣系统（DnaK- DnaJ-GrpE）和/或折叠催化剂（触发因子）提高了大肠杆菌细胞中产生的重组酶的溶解度。比活性，与胺基的亲和力纯化的可溶性重组酶对钙激活和GTP抑制的敏感性要低于天然肝酶。这些结果表明折叠调节剂测试的共过量表达提高了过量生产的重组哺乳动物组织类型的溶解度转谷氨酰胺酶，但可溶性重组酶的催化性能与天然酶不完全相同。

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