Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA.
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.
Zhang
Zhong
Z
Academia Sinica, Beijing, China.
Song
Li-ping
LP
Fang
Min
M
Wang
Fei
F
He
Dan
D
Zhao
Rui
R
Liu
Jing
J
Zhou
Zhi-yong
ZY
Yin
Chang-cheng
CC
Lin
Qing
Q
Huang
Hua-liang
HL
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
D000906:Antibodies; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli