新型双功能谷胱甘肽合成酶的真核和原核表达

通过裂解无乳链球菌(Streptococcus agalactiae)菌体细胞得到基因组DNA。根据GenBank上已发表的无乳链球菌(菌种编号ATCC13813)新型双功能谷胱甘肽合成酶基因(gshF)的核酸序列与2种不同表达载体多克隆位点序列，分别设计引物F₁、R₁和F₂、R₂，再以总DNA为模板，经过特异性PCR扩增出长度约为2 200 bp的目的基因。随后分别对目的基因gshF和2种表达载体进行双酶切、连接等操作，得到表达载体pPIC9K-gshF与pET-gshF。用Sal I线性化表达载体pPIC9K-gshF后电转至毕赤酵母GS115中。经MD平板筛选重组子、菌落PCR鉴定阳性菌株、G418抗性梯度平板筛选多拷贝菌株，最终在4.0 mg·mL^-1 G418抗性平板上筛选出阳性菌株。用甲醇终浓度为2%的BMMY培养基诱导该阳性菌株表达，每隔12 h取样并添加甲醇，96 h后离心收集发酵上清，经SDS-PAGE蛋白电泳显示：在85 kDa处出现1条明显蛋白带，大小与预期GshF蛋白一致。重组菌BL21-pET-gshF用IPTG诱导表达，先在37 ℃下培养90 min，再加入IPTG至1 mmol·L^-1后诱导7 h，离心收集表达产物并对重组菌进行超声破碎处理后，进行SDS-PAGE蛋白电泳，同样在85 kDa处有1条蛋白带。采用Bradford法测定2种表达产物上清的蛋白量，其中重组酵母表达上清中蛋白量为0.46 mg·mL^-1，重组大肠杆菌表达产物破壁后的蛋白量为1.46 mg·mL^-1。测定并比较2种不同表达方式所得酶活，发现GshF在毕赤酵母表达中的比酶活仅为14.15 U·mL^-1，经原核表达后比酶活可达62.15 U·mL^-1。

Expression of Novel Bifunctional Glutathione Synthetase in Pichia pastoris and Escherichia coli

Genomic DNA can be obtained by lysis of Streptococcus agalactiae. According to the nucleic acid sequence of (GshF) published on the Genbank (strain code: ATCC13813) and two different expression sequences, primers F₁, R₁ and F_2, R₂ can be designed. Using genomic DNA as template, the target gene gshF with a length of about 2 200 bp was amplified by specific PCR. The recombinant expression vector pPIC9K-gshF and pET-gshF were constructed by inserting into Pichia pastoris and Escherichia coli through double enzyme digestion. After Sal I linearized pPIC9K-gshF, electro transforming into GS115. The recombinant strains were screened by MD plate, identified by colony PCR and screened by G418 resistance gradient plate. Finally, the positive strains with G418 resistance of 4.0 mg·mL^-1 were screened. The positive strain was induced by BMMY medium with 2% final methanol concentration. Samples were taken every 12 h and methanol was added. The fermentation supernatant was collected by centrifugation after 96 h SDS-PAGE electrophoretic band analysis and Bradford protein content determination were carried out. The SDS polyacrylamide gel electrophoresis showed that there was a protein band in the recombinant strain(GS115-pPIC9K-gshF), which was consistent with the target protein size at 85 kDa. Meanwhile the expression of recombinant strain BL21-pET-gshF was directly induced by IPTG. After incubation at 37 ℃ for 90 min, IPTG was added to 1 mmol·L^-1 and incubated for 7 h. The expression product was treated by ultrasonic cell breaking, also SDS-PAGE electrophoretic band analysis and Bradford protein content determination were carried out. There was also a band at 85 KDa. The results show that GshF is more suitable for expression in E.coli, and the activity of GshF is 62.15 U·mL^-1, but only 14.15 U·mL^-1 in Pichia pastoris.