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Updated: Aug 16, 2026
Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a
Sedighe Kolivand1, Mahboobeh Nazari2, Mohammad Hossein Modarressi3
1Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Background:
Inhibin A, a member of TGF-β superfamily, consists of α and β subunits. These subunits contain several cysteine residues in amino acid sequence that forms inter- and intra-subunits disulfide bonds. Due to the reducing environment of the bacterial cytoplasm, disulfide bonds formation in E.coli cytoplasm is not possible. Therefore, this can cause misfolding, aggregation and inclusion bodies formation during protein expression. As a result, the expression of inhibin subunits in E.coli produces inclusion bodiesOBJECTIVE: We aimed at identification of an optimized protocol for expression and recovery of inhibin α-subunit from inclusion bodies.
Methods:
Two vectors, four different E.coli strains, and six solubilization conditions for were used for the optimization of inhibin α-subunit production. Then, the solubilized proteins were purified through Ni-NTA affinity chromatography, characterized by SDS-PAGE and Western blotting (WB) using anti-his tag antibody, and refolded by dilution.
Results:
The results showed that inhibin α-subunits were successfully expressed in both vectors and the pET22b+inhibin α-subunit in ShuffleTM T7 strain had the highest expression; however, most of the expression was in an insoluble form. Among solubilization buffers examined, a buffer containing 2M urea with pH 12 was the best buffer to dissolve the insoluble protein. The high purity of protein was confirmed by SDS-PAGE and WB. Non-reducing SDS-PAGE demonstrating inhibin α-subunit refolded well.
Conclusion:
The current protocol is an efficient method for protocol for expression and recovery of inhibin α-subunit from inclusion bodies.
