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To investigate the structure-function relationship of the pro-region in mediating tissue-specific cleavage of peptide hormone precursors, prosomatostatins (proSRIFs) from anglerfish were overexpressed in F_ colt and purified. cDNAs of preproSRIF I and H genes from anglerfish Lophius americanus were inserted into the F_ coli glutathione S-transferase (GST) fusion vector, pGEX-3X, and periplasmic secretion vector, pIN-ffl-ompA2, respectively. By oGgonucleotidedirected mutagenesis using polymerase chain reaction (PCR), linker region, 5-untranslated sequence, and signal peptide were deleted. When cells harboring these constructs were induced with 1 mM IPTG, GST-proSRIF I of 38 kDa and proSRIF H of 14 kDa proteins were overproduced. ProSRIF I protein fused to GST was purified by affinity chromatography using glutathione Sepharose 4B, followed by cleavage of GST fusion protein with activated factor X. ProSRIF U protein was purified first by collecting the periplasmic fraction after osmotic shock and then through the conventional column chromatography methods. These purified pro-hormone peptides will be used to crystallize and determine their tertiary structures.
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