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
References
Aug 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·J S HustonR Crea
Aug 15, 1970·Nature·U K Laemmli
Mar 6, 1998·Trends in Biotechnology·G Hannig, S C Makrides
Nov 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·P H BessetteG Georgiou
Oct 26, 2001·Protein Expression and Purification·R LevyG Georgiou
Jun 13, 2003·Journal of Biochemistry·Lei ZhengJean-Louis Reymond
Jan 23, 2004·Journal of Immunological Methods·Ville Santala, Urpo Lamminmäki
Oct 7, 2004·Current Opinion in Biotechnology·Dana C Andersen, Dorothea E Reilly
Jan 18, 2006·Protein Expression and Purification·Mi-Ae HeoSun-Gu Lee
Jan 24, 2006·Journal of Molecular Biology·Paola JuradoLuis A Fernández
Sep 29, 2006·Protein Expression and Purification·Xuejun HuJ Gerard Wall
May 3, 2008·Proteomics·Shannon L Seurynck-ServossRichard C Zangar
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 14, 2014·BioMed Research International·Georg HuberHeribert Stoiber
Feb 4, 2014·Antioxidants & Redox Signaling·Marizela DelicBrigitte Gasser
Apr 20, 2011·Journal of Immunological Methods·Dominique DesplancqEtienne Weiss
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
Jan 17, 2012·Protein Expression and Purification·Ganesh P SubediYoshiki Yamaguchi
Sep 4, 2012·Bioresource Technology·Kamna Jhamb, Debendra K Sahoo
Apr 30, 2013·Journal of Immunological Methods·Raphael LevyToshihiko Takeuchi
Aug 15, 2014·Biochimica Et Biophysica Acta·Yoichi Kumada
Oct 8, 2016·PloS One·Ondřej KaplanEduard Brynda
Jun 11, 2017·Virus Genes·Yu ZhangAnchun Cheng
Sep 24, 2019·Antibodies·Qiong WangMichael J Betenbaugh
Sep 4, 2020·International Journal of Molecular Sciences·Annamaria SandomenicoMenotti Ruvo