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Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface
V Akbari1, H Mir Mohammad Sadeghi1, A Jafarian-Dehkordi1
1Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
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.
Insights
Researchers optimized the expression of anti-HER2 single-chain variable fragments (ScFv) in E. coli. Guanidine hydrochloride proved more effective than urea for solubilizing inclusion bodies, achieving 95% purity.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- The Human Epidermal growth factor Receptor (HER) family is crucial in cancer development.
- Antibodies targeting HER receptors, like HER2, are vital for cancer therapy.
- Previous work established an anti-HER2 single-chain variable fragment (ScFv) for Escherichia coli expression.
Purpose of the Study:
- To optimize the expression of anti-HER2 ScFv in E. coli BL21 (DE3) pLysS.
- To identify optimal cultivation conditions for maximizing anti-HER2 ScFv yield.
- To evaluate purification strategies for anti-HER2 ScFv.
Main Methods:
- Response surface methodology (Box-Behnken design) was employed to optimize expression.
- Cultivation variables optimized included IPTG concentration, temperature, and post-induction time.
- Inclusion body isolation and Ni-NTA affinity chromatography were used for purification.
Main Results:
- A predictive model indicated maximal anti-HER2 ScFv expression at 37 °C, 10.45 h post-induction, and 0.75 mM IPTG.
- Purification via Ni-NTA resin yielded a final product with 95% purity.
- Guanidine hydrochloride demonstrated superior efficacy over urea in solubilizing inclusion bodies.
Conclusions:
- Optimized conditions significantly enhance anti-HER2 ScFv expression in E. coli.
- High-purity anti-HER2 ScFv can be obtained using inclusion body isolation and affinity chromatography.
- Guanidine hydrochloride is the preferred agent for solubilizing anti-HER2 ScFv inclusion bodies.
