Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a cysteine rich peptide chain.

Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a cysteine rich peptide chain. Hum Antibodies. 2019 Oct 18;: Authors: Kolivand S, Nazari M, Modarressi MH, Najafabadi MRH, Hemati A, Ghafouri-Fard S, Motevaseli E Abstract 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. METHOODS: 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 ShuffleT...
Source: Human Antibodies - Category: Biochemistry Tags: Hum Antibodies Source Type: research