title: Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli.
abstract: OBJECTIVE
  Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody.
  
  
  METHODS
  To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively.
  
  
  RESULTS
  SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively.
  
  
  CONCLUSIONS
  The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. ; Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Kamal
authors.initials: K
authors.lastname: Veisi
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Safar
authors.initials: S
authors.lastname: Farajnia
authors.affiliation: Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Nosratollah
authors.initials: N
authors.lastname: Zarghami
authors.affiliation: University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Khoram Khorshid
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Nasser
authors.initials: N
authors.lastname: Samadi
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Shiva
authors.initials: S
authors.lastname: Ahdi Khosroshahi
authors.affiliation: Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Zarei Jaliani
keywords: EGFR; Molecular chaperones; ScFv; Soluble expression