Zhang Zhong, Song Li-ping, Fang Min, Wang Fei, He Dan, Zhao Rui, Liu Jing, Zhou Zhi-yong, Yin Chang-cheng, Lin Qing, Huang Hua-liang
Academia Sinica, Beijing, China.
Biotechniques. 2003 Nov;35(5):1032-8, 1041-2. doi: 10.2144/03355rr03.
Overproduction of genetically engineered antibodies, such as single-chain antibodies (scAbs) in Escherichia coli often results in insoluble and inactive products known as inclusion bodies. We now report that fusion or co-expression of FkpA, the E. coli periplasmic peptidyl-prolyl-isomerase with chaperone activity, substantially improves soluble and functional expression of scAbs. Anti-human bladder carcinoma scAb (PG) and anti-human CD3 x anti-human ovarian carcinoma-bispecific scAb (BH1) were fused with FkpA on the pTMF-based plasmid and expressed in E. coli. More than half of the amount of each expressed fusion protein FkpA-PG or FkpA-BH1 was soluble. In addition, the fusion protein cellulose-binding domain from Cellulomonas fimi (CBD)-PG and anti-human CD3 x anti-human CD28 x anti-human ovarian carcinoma-trispecific scAb (TRI) fused to the pelB (a signal peptide from pectate lysase B of a Bacillus sp.) signal sequence were co-expressed with FkpA under the control of the T7 promoter. A substantial portion of the co-expressed CBD-PG or TRI was soluble. Furthermore, PG, BH1, and TRI were biologically active as judged by ELISA and in vitro cytotoxicity assay. These results suggest that overexpression of FkpA should be useful in expressing heterologous proteins in E. coli.
在大肠杆菌中过量生产基因工程抗体，如单链抗体（scAbs），常常会产生不溶性且无活性的产物，即包涵体。我们现在报道，将具有伴侣活性的大肠杆菌周质肽基脯氨酰异构酶FkpA进行融合或共表达，可显著提高scAbs的可溶性和功能性表达。抗人膀胱癌scAb（PG）和抗人CD3×抗人卵巢癌双特异性scAb（BH1）在基于pTMF的质粒上与FkpA融合，并在大肠杆菌中表达。每种表达的融合蛋白FkpA-PG或FkpA-BH1超过一半的量是可溶的。此外，来自纤维单胞菌的融合蛋白纤维素结合结构域（CBD）-PG和与pelB（芽孢杆菌属果胶酸裂解酶B的信号肽）信号序列融合的抗人CD3×抗人CD28×抗人卵巢癌三特异性scAb（TRI）在T7启动子的控制下与FkpA共表达。共表达的CBD-PG或TRI的很大一部分是可溶的。此外，通过ELISA和体外细胞毒性试验判断，PG、BH1和TRI具有生物学活性。这些结果表明，FkpA的过表达在大肠杆菌中表达异源蛋白方面应该是有用的。
