Practice question

Human growth hormone somatotropin monomeric 22 kDa protein 191 aa two intramolecular disulfide bridges Cys53-Cys165 Cys182-Cys189 secreted somatotrophs anterior pituitary under GHRH stimulates hepatic IGF-1 production promoting longitudinal bone growth protein synthesis. Recombinant production first accomplished E. coli K12 because hormone lacks N-linked glycosylation essential in vivo activity unlike erythropoietin requiring sialylated glycans circulatory half-life, making prokaryotic expression feasible cost-effective. Gene without native signal peptide inserted downstream tryptophan operon promoter trp or hybrid tac induced 3-beta-indoleacrylic acid depletion isopropyl beta-D-thiogalactoside synthesizing inclusion bodies accumulating up to 30 percent total cellular protein. Inclusion bodies denatured 6 M guanidine HCl dithiothreitol reducing disulfides then refolded stepwise dialysis against arginine buffer pH 8.5 allowing correct disulfide pairing via air oxidation. Purification DEAE Sepharose anion exchange Sephadex G100 gel filtration yielded monomer N-terminal methionine retained early product somatrem Protropin associated antibody formation, later somatropin produced via aminopeptidase removal. Bacterial fermentation scaled 10000 L capacity meeting global pediatric deficiency demand low cost decades.