Functional expression of single-chain Fv antibody in the cytoplasm of Escherichia coli by thioredoxin fusion and co-expression of molecular chaperones

Protein Expression and Purification
Hiroyuki SonodaHideki Yamaji

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...Continue Reading

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Citations

Sep 9, 2011·Journal of Industrial Microbiology & Biotechnology·Hendrik Waegeman, Wim Soetaert
Oct 29, 2013·Microbial Cell Factories·Kristof ZarschlerHolger Stephan
May 8, 2014·PloS One·Gianluca PetrisOscar R Burrone
Feb 18, 2011·Journal of Bioscience and Bioengineering·Hiroyuki SonodaHideki Yamaji
Apr 29, 2015·Journal of Bioscience and Bioengineering·Yong Jae Lee, Ki Jun Jeong
Dec 21, 2010·Biotechnology Journal·Ki Jun JeongNatarajan Velmurugan
May 11, 2015·The International Journal of Biochemistry & Cell Biology·Megha Goyal, Tapan K Chaudhuri
Sep 24, 2019·Antibodies·Qiong WangMichael J Betenbaugh
Sep 4, 2020·International Journal of Molecular Sciences·Annamaria SandomenicoMenotti Ruvo

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