title: Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology.
abstract: Human epidermal growth factor receptor (HER) family plays an important role in various types of cancers. As a result, antibodies against HER and the mechanism of antigen-antibody binding action are under active investigation. We previously constructed a single-chain variable fragment (ScFv) against HER2, i.e. anti-Her2 ScFv, for expressing in the Escherichia coli. In the present study, we report the optimization of anti-Her2 ScFv expression in an E. coli host of BL21 (DE3) pLysS using response surface methodology based on tuning of three cultivation variables, including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. A model for protein expression according to the Box-Behnken design predicted a maximal anti-Her2 ScFv expression at 37 °C, a post-induction time of 10.45 h and 0.75 mM IPTG. In addition, strategies based on inclusion body isolation and affinity chromatography were applied to purify anti-Her2 ScFv. The purity of the final product for inclusion bodies isolation and purification by Ni-NTA resin were 70 % and 95 %, respectively. The solubilization of the inclusion bodies was carried out using two denaturant agents, guanidine hydrochloride and urea. The present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies.
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: V
authors.initials: V
authors.lastname: Akbari
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: H Mir Mohammad
authors.initials: HM
authors.lastname: Sadeghi
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: A
authors.initials: A
authors.lastname: Jafarian-Dehkordi
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: C Perry
authors.initials: CP
authors.lastname: Chou
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: D
authors.initials: D
authors.lastname: Abedi
keywords: Antibody; Escherichia coli; Human epidermal growth factor receptor (HER); Response surface methodology; Single-chain variable fragment (ScFv)