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Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2010 Apr; Vol. 70 (2), pp. 248-53. Date of Electronic Publication: 2009 Nov 12. - Publication Year :
- 2010
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Abstract
- 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.<br /> ((c) 2009 Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Antigen-Antibody Reactions
Cattle
Chaperonin 60 biosynthesis
Cytoplasm metabolism
Drug Stability
Escherichia coli Proteins biosynthesis
Hot Temperature
Hydrogen-Ion Concentration
Peptidylprolyl Isomerase biosynthesis
Recombinant Fusion Proteins genetics
Ribonuclease, Pancreatic immunology
Escherichia coli metabolism
Immunoglobulin Variable Region biosynthesis
Molecular Chaperones genetics
Recombinant Fusion Proteins biosynthesis
Single-Chain Antibodies biosynthesis
Thioredoxins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 70
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 19913620
- Full Text :
- https://doi.org/10.1016/j.pep.2009.11.003