Practice question

Somatostatin tetradecapeptide 14 aa sequence Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys cyclized via disulfide cysteines 3 and 14 functions potent inhibitor growth hormone insulin glucagon secretion, making direct bacterial expression challenging small bioactive peptide inserts membrane disrupts host metabolism undergoes rapid proteolysis cytoplasmic peptidases OmpT. To overcome instability Itakura 1977 expressed synthetic DNA encoding somatostatin fused downstream E. coli beta-galactosidase fragment 590 residues or tryptophan operon leader peptide trpE 190 residues under trp promoter regulated tryptophan repression generating larger chimera lacking hormonal activity sequestered insoluble inclusion bodies resistant proteolytic attack. Fusion design included methionine codon immediately preceding somatostatin enabling specific chemical cleavage CNBr releasing free tetradecapeptide after protection internal methionines absent. Following cleavage peptide extracted 50 percent acetic acid purified ion exchange CM cellulose reversed-phase HPLC oxidized ferricyanide forming disulfide loop essential binding somatostatin receptors SSTR2 SSTR5. Pioneering fusion approach established general paradigm manufacturing small peptides neurohormones industrially influencing insulin opioid peptides.