Epinephelus coioides growth hormone cDNA cloning and fusion expression in Escherichia coli

Epinephelus coioides GH cDNA was cloned from pituitary cDNA library by random sequencing. The sequence obtained spanned 955bp, w ith an open reading frame encoding a protein of 204 amino acids, which is composed of a putative signal peptide of 17 residues and a mature polypeptide of 187 amino acids. The cDNA f ragment encoding the mature polypeptide of GH was PCR amplif ied and subcloned to expression vector pET- 15b ( Novagen) , and expressed in E. coli BL21 ( DE3) as fusion polypept ide containing a His6 at the N - terminus. The addit ion of 0. 4 mmol..L - 1 IPTG induced expression of a protein band with molecular weight of about 24 kDa. The expressed protein accumulated as inclusion bodies, which were solubilized in 6 mol..L- 1 guanidine HCl, and further purif ied and renatured on Ni2 - NTA resin. The purif ied Epinephelus coioides GH fusion polypeptide migrated as a single band of 24 kDa on SDS- PAGE and exhibited GH immunoreactivity in sea bream GH RIA system.