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-beta-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 amino-terminal analysis showed the purified protein to be monomeric methionyl-dfGH. Binding assays of the (125)I-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 EC(50) values were 28 nM versus 0.095 nM.
Paduel
A
A
National Institute of Oceanography, Israel Oceanographic & Limnological Research, in Eilat, Eilat, 88112, Israel.
Chapnik-Cohen
N
N
Gertler
A
A
Elizur
A
A
D000818:Animals; D002347:Carps / Q000378:metabolism; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D013006:Growth Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010473:Perciformes / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D008565:Membrane Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone