色氨酸酶在大肠杆菌周质中的分泌融合表达

Secretory and fusion expression of trypt-ophanase gene in the periplasmic space of Escherichia coli

按照大肠杆菌偏爱密码子合成编码"BspTI位点-连接肽(Gly-Ser-Gly-Ser-Gly-6His-Tag-Ek-site(Asp-Asp-Asp-Asp-Lys)"序列的DNA序列,利用重叠延伸拼接技术,将其与色氨酸酶基因TnaA拼接,利用BspTI和HindⅢ重组至pET-39二硫键形成蛋白DsbA基因的下游,转化表达宿主Escherichia coliBL21(DE3),成功构建分泌融合表达型色氨酸酶基因工程菌.通过摇瓶培养实验研究了诱导剂浓度、诱导温度及诱导时间对色氨酸酶活性的影响.工程菌以含2%甘油的LB为培养基,A600=0.5时,加入终浓度为0.05 mmol/L的Isopropyl-βD-1-thiogalactopyranoside(IPTG),30℃诱导培养8 h,可在周质空间中表达高活性的色氨酸酶融合蛋白.在培养基中添加0.5%的甘氨酸,酶活可提高6.3倍.本实验首次实现了色氨酸酶基因的周质分泌融合表达.

The gene encoding BspTI site-linker peptide （Gly-Ser-Gly-Ser-Gly-6His-Tag-Eksite （Asp-Asp-Asp-Asp-Lys） was synthesized according to the preferential codons of Escherichia coli, and tryptophanase gcne were spliced by overlap extension . Then the target gene was coloned to the downstream of disulfide bond formation protein a gene of pET-39 and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3）. The engineering strain for secretory expression TnaA fusion protein was successfully constructed. The effects of concentration of Isopropyl β-D-1- thiogalactopyranoside （IPTG）, induction temperature and induction time on the enzyme activity were studied with shaking flask method. The optional expression condition in the LB medium containing 2 % glycerol was that the induction started as A600 reached about 0.5 by adding IPTG to a final concentration of 0.05 mmol/L for 8 hours at 30 ℃. Moreover, about 6.3-fold increase in the activity of tryptophanase could be obtained by addition of 0. 5% glycine to the culture medium . This is the first secretory expression of tryptophanase gene in E. coli