茂原链霉菌谷氨酰胺转胺酶基因克隆、序列分析及原核表达

Cloning,Sequence Analysis and Expression of Transglutaminase Gene from Streptomyces mobaraensis

以茂原链霉菌(Streptomyces m obaraensis)的基因组DNA为模板,利用PCR的方法扩增出谷氨酰胺转胺酶(Transglutam inase,TGase)的完整基因片段.序列分析结果表明,该片段全长1 246 bp,包含一个完整的ORF,长度1 146 bp,编码395 aa,分子量为44 kD左右.该基因的核酸序列及推导的氨基酸序列同已知微生物来源的若干TGase相比,序列相似性均很高,并且发现与酶催化活性有关的一些motif,如酰胺化位点等;在此基础上,又进一步对其二级结构进行分析并完成了TGase三维结构的建模.将编码TGase酶的基因片段插入到原核表达载体pQE30Xa中,并转化大肠杆菌(E.coliJM109).SDS-PAGE结果表明,经IPTG诱导后该基因得到表达,表达产物的酶活为2.2 U/mL.

Transglutaminase （ TGase ; protein-glutamine ： amine γ-glutamyltransferase, EC 2.3.2.13 ） catalyzes an acyl transfer reaction between aγ-carboxyamide group of glutamine and ε-amino group of lysine or other primary amine, resulting in the formation of γ-glutamyl-ε-lysine peptide chain bridges. A gene, encoding TGase, 1 246 bp length in Streptomyces moboransis was cloned by PCR（ polymerase chain reaction）. Analysis of TGase gene sequence showed that the sequence was just a complete ORF, 1 146 bp length, encoded a protein with 395 amino acids, 44 kD molecular weight. It had higher similarity with those TGase reported from other microorganisms. Besides, TGase form Streptomyces moboransis contained some important motifs related with enzyme activity such as amidation site, N-glyeosylation site etc. Based on motifs prediction, secondary structure was predicted and three-dimensional structure of TGase was developed by homology modeling methods, which discovered the 125^th amino acid cysteine as enzyme active site embedded into surface of TGase molecule. The gene coding TGase was inserted into pQE30xa vector and was transformed into E. coli JM109 strain. After induced by IPTG, the result of SDS-PAGE screened that the TGase was expressed, enzyme activity 2.2 U/mL. This suggests that the TGase gene can be cloned and expressed in Escherichia coli. Once recombined TGase obtained efficient expression and enzyme activity, it will be fully possible to produce TGase by industrialization