Mohammad Sina
1,2, Davoud Farajzadeh
2,3, Siavoush Dastmalchi
2,4*1 Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3 Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran.
4 Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
Purpose: The bacterial
cultivation conditions for obtaining anti-TNF-α single chain variable fragment
(scFv) antibody as the soluble product in E. coli was investigated.
Methods: To avoid the
production of inclusion bodies, the effects of lactose, IPTG, incubation time,
temperature, shaking protocol, medium additives (Mg+2, sucrose), pH,
osmotic and heat shocks were examined. Samples from bacterial growth conditions
with promising results of soluble expression of GST-hD2 scFv were affinity
purified and quantified by SDS-PAGE and image processing for further
evaluation.
Results: The results showed
that cultivation in LB medium under induction by low concentrations of lactose
and incubation at 10 °C led to partial solubilization of the expressed
anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in
soluble expression of GST-hD2 were osmotic shock and addition of magnesium
chloride. Furthermore, addition of sucrose to medium suppressed the expression
of scFv completely. The other finding was that the addition of sorbitol
decreased the growth rate of bacteria.
Conclusion: It can be concluded that low cultivation temperature in the presence of low
amount of inducer under a long incubation time or addition of magnesium
chloride are the most effective environmental factors studied for obtaining the
maximum solubilization of GST-hD2 recombinant protein.