酵母表达长角血蜱谷氨酰胺转移酶及其活性分析
首发时间:2016-06-24
摘要:谷氨酰胺转移酶(Transglutaminase:EC2.3.2.13,简称为TGase)通过催化蛋白质的谷氨酰胺残基与赖氨酸残基之间形成ε-(γ-谷氨酰基)赖氨酸异肽键,或在与肽结合的谷氨酰胺残基处掺入伯胺,促进蛋白分子内和分子间的交联,形成网状的高分子聚合物,参与多种重要的生物学活动。为评估该分子在长角血蜱中的作用及其可能的应用前景,从长角血蜱(Haemaphysalis longicornis)上海株中克隆了谷氨酰胺转移酶基因HlTGase(GenBank KX59300),并将其重组表达在毕赤酵母中,进而对其编码蛋白的分子特征及可能的应用价值进行了评估。结果显示:该基因的开放阅读框为2262bp,编码了一条756aa的多肽链,该多肽链具有四个谷氨酰胺转移酶结构域,理论分子量为84.6kDa;系统发育树显示其与果蝇的谷氨酰胺转胺酶亲缘关系最近;在毕赤酵母中成功表达的重组蛋白具有谷氨酰胺转移酶的活性,能催化酪蛋白交联成较大的分子,具有一定的应用前景。
关键词: 分子特征 长角血蜱 谷氨酰胺转移酶 催化活性 毕赤酵母
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Analysis on a transglutaminase from Haemaphysalis longicornis expressed in Pichia pastoris
Abstract:Transglutaminases (TGases) are a widely distributed group of enzymes that catalyze the formation of isopeptide bonds either through protein cross-linking via ε-(γ-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide- bound glutamine residues, and involved in multiple important physiological events. In this paper, a transglutaminase gene was cloned from a vector tick, Haemaphysalis longicornis Shanghai strain (HlTGase, GenBank accession number: KX59300), and expressed in Pichia pastoris successfully. The molecular characterization and the enzyme activity of the recombinant HlTGase were analyzed. The result showed that the open reading frame of the gene was 2262 bp, which encoded a polypeptide of 756 aa. The polypeptide possessed four transglutaminase domains. The calculated molecular weight of the polypeptide was 84.6 kDa. A phylogenetic tree showed that the polypeptide had a closest relationship with that of Drosophila melanogaster.among TGases from 11 typical species, which was correspondent with the traditional taxonomical status. The antibody against the recombinant HlTGase recognized the endogenous HlTGase in a western blotting analysis. The results also showed that the recombinant HlTGase had enzyme activity to catalyze cross linking between proteins. However, the activity was lower than a commercial available TGase from guinea pigs. More modification in the expression and purification of the recombinant HlTGase might be required to improve the enzyme activity. This study would provide basic information for further study on the function and potential application of HlTGase.
Keywords: molecular characterization Haemaphysalis longicornis transglutaminase enzyme activity Pichia pastoris
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