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 Es cherichia coli BL21 cells upon induction with isopropyl-1-thio-beta-D-galac topyranoside as an insoluble protein. The expressed protein, contained with in 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 hom ogeneity, as evidenced by SDS-PAGE. Gel filtration on a Superdex column und er nondenaturing conditions and aminoterminal analysis showed the purified protein to be monomeric methionyl-dfGH. Binding assays of the I-125-labeled dfGH to dolphin fish liver microsomal fraction resulted in high specific b inding characterized by a K-a of 0.77 nM(-1) and a B-max of 285 fmol/mg mic rosomal fraction protein. The purified dfGH was capable of stimulating prol iferation of FDC-P1-B9 cells transfected with rabbit growth hormone (GH) re ceptor. The maximal effect of dfGH was identical to that of human GH but th eir respective EC50 values were 28 nM versus 0.095 nM. (C) 1999 Academic Pr ess.