Premium
Production of bubaline somatotropin by auto‐induction in Escherichia coli
Author(s) -
Sadaf Saima,
Khan Muhammad Altaf,
Akhtar Muhammad Waheed
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060154
Subject(s) - escherichia coli , chemistry , chromatography , inclusion bodies , isopropyl , affinity chromatography , cell growth , biochemistry , enzyme , organic chemistry , gene
Production of His–BbST [hexahistidine‐tagged BbST (bubaline somatotropin)] by auto‐induction in high‐density shake‐flask cultures coupled with a single‐step, on‐column purification and refolding strategy is described here. To optimize expression of BbST, different media and expression conditions were tested. The highest expression levels of BbST, exceeding 30% of the total Escherichia coli cellular proteins, were achieved when YNG and M9NG media were used. Using these auto‐inducing media, the final concentration of BbST increased up to 455 mg/l and was severalfold higher than that obtained by isopropyl β‐ d ‐thiogalactoside induction. Most of the target protein, however, was in the form of inclusion bodies, which were solubilized in 8 M urea solution (pH 8.5). Using immobilized‐metal‐ion‐affinity chromatography, His–BbST could be purified from solubilized sample to >97% homogeneity in a single step in a biologically active state as judged by its efficient growth‐promoting activity in Nb2 rat lymphoma cell proliferation assays. The expression and purification scheme, presented here, has a potential of scaling up to obtain pure and biologically active His–BbST relatively inexpensively for further studies and applications.