Production of a biologically active novel goldfish growth hormone in Escherichia coli.
Goldfish pituitary contains two types of growth hormones. One with five cysteine residues (type-I) similar to other Cyprinid GHs, and the other with four Cys residues (type-II) similar to those of other fish and tertapod species. Recombinant goldfish type II GH (gfGH-II) was produced in Escherichia coli using the pRSETB expression vector. The gfGH-II was produced fused to a leader sequence, which sequestered into inclusion bodies after expression. The inclusion bodies were solubilized using sodium hydroxide and the fusion protein purified by chelating affinity chromatography. Subsequently, gfGH-II was cleaved and analyzed by Western blotting, using a specific antiserum. For comparison we also produced recombinant common carp GH (cGH) which has 95% similarity to gfGH-II, and tested their growth promoting activity in goldfish. Both forms of GH significantly increased the growth rate of goldfish (P < 0.05), although cGH was found to have a somewhat higher potency than gfGH-II.
Mahmoud
S S
SS
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Wang
S
S
Moloney
M M
MM
Habibi
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics*; D006054:Goldfish / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D010450:Endopeptidases