Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones.
The production of a single-chain variable fragment (scFv) antibody against bovine ribonuclease A in the cytoplasm of Escherichia coli trxB/gor double mutant was investigated. Previous reports have shown that the thioredoxin (Trx) protein fusion strategy is useful for the correct folding of scFvs and that the expression of functional scFvs is increased by co-expression of molecular chaperones. In the present study, we examined the effects of the combination of Trx fusion and molecular chaperone co-expression on the production of a functional scFv. A Trx-fused scFv was obtained in the oxidizing cytoplasm, and co-expression of GroELS and trigger factor had the greatest effect, resulting in a 2.8-fold increase in specific productivity. By contrast, the molecular chaperone DnaKJE had no effect. Moreover, co-expression of DnaKJE with GroELS negated the effects of GroELS. Trx-scFv was purified using a bovine ribonuclease A-coupled Sepharose column, and 2.7 mg/L of purified protein was obtained. Soluble Trx-scFv, expressed and purified as described above, exhibited pH-dependent binding similar to that of the parental full-length antibody. In addition, approximately 80% of the initial binding activity was retained after incubation at 37 degrees C for 2 weeks, indicating that the Trx-scFv fusion protein is quite stable. This strategy might be useful for the preparation of other recombinant scFvs.
Sonoda
Hiroyuki
H
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Kumada
Yoichi
Y
Katsuda
Tomohisa
T
Yamaji
Hideki
H
D000818:Animals; D000937:Antigen-Antibody Reactions; D002417:Cattle; D018834:Chaperonin 60 / Q000096:biosynthesis; D003593:Cytoplasm / Q000378:metabolism; D004355:Drug Stability; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D018832:Molecular Chaperones / Q000235:genetics*; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000276:immunology; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D013879:Thioredoxins / Q000235:genetics*
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D013879:Thioredoxins; D012259:Ribonuclease, Pancreatic; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase