Test bacterial inclusion body for activity prior to start denaturing and refolding processes to obtain active eukaryotic proteins
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Seyedeh Roghayeh Hamidi,1 Yaghoub Safdari,2,* Mehdi Sheikh-Arabi,3
1. Department of Medical Biotechnology, Faculty of Advanced Technologies in Medicine, Golestan University of Medical Sciences,
2. Golestan Research Center of Gastroenterology and Hepatology, Golestan University of Medical Sciences
3. Medical cellular and molecular research center, Golestan University of Medical Sciences
- Introduction: One of a major drawbacks correlated with expressing antibody fragments in bacterial cells is insolubility, which is often regarded as an obstacle in obtaining active molecules. Recombinant proteins aggregated as inclusion bodies within bacterial cells are thought to be unfolded or misfolded, and therefore inactive. So, denaturing and refolding strategies, which are laborious and sometime inefficient, are used to obtain correctly-folded active proteins. In the current study, we show that large quantities of correctly folded and completely active scFv molecules are there in bacterial inclusion bodies; they only need to be isolated from inclusion bodies.
- Methods: Method
One of a major drawbacks correlated with expressing antibody fragments in bacterial cells is insolubility, which is often regarded as an obstacle in obtaining active molecules. Recombinant proteins aggregated as inclusion bodies within bacterial cells are thought to be unfolded or misfolded, and therefore inactive. So, denaturing and refolding strategies, which are laborious and sometime inefficient, are used to obtain correctly-folded active proteins. In the current study, we show that large quantities of correctly folded and completely active scFv molecules are there in bacterial inclusion bodies; they only need to be isolated from inclusion bodies.
- Results: Result
The gene encoding 9R-nimotuzumab scFv was synthesized and subcloned into a pET22b(+) vector. The recombinant plasmid was transformed into E. coli BL21(DE3) and the fusion protein overexpressed in bacterial cells under IPTG induction. After lysing cells,, the pellet which contained inclusion body was dissolved in Tris solution without denaturation agents. The recovered protein was evaluated with ELISA assay.
- Conclusion: Conclusion
ScFv molecules expressed in bacterial cells as inclusion body may be correctly folded and therefore be active. It is better to solubilize inclusion bodies with a non-denaturing buffer and evaluate the activity of recovered protein prior to starting time-consuming, and sometimes
inefficient, denaturing and refolding protocols.
- Keywords: ScFv, Inclusion body, Native 3D structure, Refolding, Non-denaturing condition
