Kamal Veisi1, Safar Farajnia2, Nosratollah Zarghami3, Hamid Reza Khoram Khorshid4, Nasser Samadi5, Shiva Ahdi Khosroshahi6, Hossein Zarei Jaliani7. 1. 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. 2. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Department of Medical Biotechnologies, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran. 4. University of Social Welfare and Rehabilitation Sciences, Tehran, Iran. 5. 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. 6. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 7. Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Abstract
PURPOSE: 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. CONCLUSION: 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.
PURPOSE: 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. CONCLUSION: 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: W Wp Rodrigo; R S Dassanayake; E H Karunanayake; Y I N Silva Gunawardene; O Vds J Weerasena Journal: Asian Pac J Trop Med Date: 2014-02 Impact factor: 1.226
Authors: Jitka Folwarczna; Tomas Moravec; Helena Plchova; Hana Hoffmeisterova; Noemi Cerovska Journal: Protein Expr Purif Date: 2012-07-28 Impact factor: 1.650