title: Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA.
abstract: 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.
authors: Academia Sinica, Beijing, China.
authors: Zhong
authors: Z
authors: Zhang
authors: Li-ping
authors: LP
authors: Song
authors: Min
authors: M
authors: Fang
authors: Fei
authors: F
authors: Wang
authors: Dan
authors: D
authors: He
authors: Rui
authors: R
authors: Zhao
authors: Jing
authors: J
authors: Liu
authors: Zhi-yong
authors: ZY
authors: Zhou
authors: Chang-cheng
authors: CC
authors: Yin
authors: Qing
authors: Q
authors: Lin
authors: Hua-liang
authors: HL
authors: Huang
mesh_terms: D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D000917:Antibody Formation / Q000235:genetics*; D002277:Carcinoma / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D020104:Immunophilins / Q000096:biosynthesis* / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D000906:Antibodies; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli