Preparation and characterization of recombinant dolphin fish (Coryphaena hippurus) growth hormone

Dolphin fish (Coryphaena hippurus) growth hormone (dfGH) cDNA encoding the mature protein was cloned in a pET11a expression vector and expressed in Escherichia coli BL21 cells upon induction with isopropyl-1-thio-β-D-galactopyranoside as an insoluble protein. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to homogeneity, as evidenced by SDS-PAGE. Gel filtration on a Superdex column under nondenaturing conditions and aminoterminal analysis showed the purified protein to be monomeric methionyl-dfGH. Binding assays of the ¹²⁵I-labeled dfGH to dolphin fish liver microsomal fraction resulted in high specific binding characterized by a K(a) of 0.77 nM^-1 and a B(max) of 285 fmol/mg microsomal fraction protein. The purified dfGH was capable of stimulating proliferation of FDC-P1-B9 cells transfected with rabbit growth hormone (GH) receptor. The maximal effect of dfGH was identical to that of human GH but their respective EC⁵⁰ values were 28 nM versus 0.095 nM.