Production of bubaline somatotropin by auto-induction in Escherichia coli.
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 beta-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.
Sadaf
Saima
S
School of Biological Sciences, University of the Punjab, Lahore, Pakistan. sasadaf9@yahoo.com
Khan
Muhammad Altaf
MA
Akhtar
Muhammad Waheed
MW
D000818:Animals; D002020:Buffaloes; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D014169:Transformation, Bacterial
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013006:Growth Hormone