Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

Identification of the gene encoding transglutaminase zymogen from Streptomyces hygroscopicus and its expression in Escherichia coli

摘要:

【目的】鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因; 研究其在大肠杆菌系统的克隆与表达; 分析该酶与其同源酶的活性中心氨基酸序列。【方法】从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus; CCTCC M203062)发酵液中, 分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase), 测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物, 扩增得到pro-MTGase基因, 将该基因插入到表达载体pET-20b(+)信号肽pelB下游, 构建分泌型表达载体pET/pro-MTG, 并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS。【结果】获得了pro-MTGase的完整基因序列, 多重碱基序列比对表明其与S. platensis和S. caniferus的pro-MTGase基因同源性高达92%。利用Rosetta(DE3)pLysS通过降温至24℃诱导策略, 获得部分胞外表达的酶原。SDS-PAGE显示, 胞外表达重组蛋白的分子量约为44kDa, 与吸水链霉菌表达的天然酶原相符。诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL。【结论】该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道, 也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达。

Abstract:

[Objective] We identified a microbial transglutaminase (MTGase) gene from Streptomyces hygroscopicus; cloned and expressed it in Escherichia coli. We also analyzed the active sites sequence of S. hygroscopicus MTGase through homologous sequence comparison. [Methods] Wild-type microbial transglutaminase zymogen (pro-MTGase) was purified from liquid culture of S. hygroscopicus(CCTCC M203062)。N-terminal amino acid sequence of this pro-MTGase was determined. According to the N-terminal sequence and the corresponding nucleotide sequence of MTGase from other three Streptomyces species, PCR primers of S. hygroscopicus pro-MTGase were designed and the completed gene of pro-MTGase was amplified and sequenced. The gene was sub-cloned into pET-20b(+) vector downstream pelB signal peptide to construct the expression vector pET/pro-MTG. [Results] The nucleotide sequence showed 92 % homologue with that of S. platensis and S. caniferus. Rosetta(DE3)pLysS carrying the expression vector was induced with IPTG at 24℃ and expressed pro-MTGase as extracellular soluble protein. SDS-PAGE showed the expressed recombinant pro-MTGase was about 44 kDa, similar to the wild-type pro-MTGase purified from S. hgroscopicus. Recombinant pro-MTGase was activated with trypsin and the enzyme activity reached to 0.24U/mL. [Conclusion] This is the first re-port of the gene encoding microbial pro-transglutaminase from S. hygroscopicus, and also this is the first report of ex-pression extracellular soluble pro-MTGase in E. coli in our country.