酵母表达的长角血蜱谷氨酰胺转移酶及其活性分析

doi:10.11843/j.issn.0366-6964.2017.02.017

摘要/Abstract

摘要：

谷氨酰胺转移酶（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，促进蛋白质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。本研究旨在克隆和表达长角血蜱（Haemaphysalis longicornis）的谷氨酰胺转移酶基因（HlTGase，GenBank登录号为KX59300），并分析重组蛋白质的活性，评估其可能的应用价值。首先从长角血蜱上海株的成虫提取总RNA，根据表达序列标签的序列信息，设计引物扩增并克隆HlTGase基因，以质粒pPICZC为表达载体，将该基因在毕赤酵母中重组表达，进而对其编码蛋白质的分子特征及可能的应用价值进行评估。结果显示HlTGase基因的开放阅读框为2 262 bp，编码了一条756 aa的多肽链，该多肽链具有四个谷氨酰胺转移酶结构域，理论相对分子质量为84.6 ku；系统发育树显示其与果蝇的谷氨酰转移酶亲缘关系最近；在毕赤酵母中成功表达的重组蛋白质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。本研究成功地用酵母表达了长角血蜱谷氨酰胺转移酶，重组蛋白质有催化蛋白质交联的活性，具有一定的应用前景。

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. This study was aimed at cloning and expression of a transglutaminase gene from a tick Haemaphysalis longicornis Shanghai strain (HlTGase, GenBank accession number: KX59300), and analysis of the molecular characterization and the enzyme activity of the recombinant HlTGase. Total RNA of the adult ticks was extracted and HlTGase gene was amplified. The open reading frame of HlTGase was inserted into a plasmid pPICZC and transformed electrically into yeast Pichia pastoris, which was then induced to express the recombinant HlTGase. The result showed that the open reading frame of the gene was 2 262 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. HlTGase was expressed in yeast successfully. 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.

中图分类号:

Q78
S852.746

胡燕红，余玲莹，黄晓红. 酵母表达的长角血蜱谷氨酰胺转移酶及其活性分析[J]. 畜牧兽医学报, 2017, 48(2): 331-339.

HU Yan-hong, YU Ling-ying, HUANG Xiao-hong. Analysis on a Transglutaminase from Haemaphysalis longicornis Expressed in Pichia pastoris[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 331-339.

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