Recombinant Carp (Cyprinus carpio) Growth Hormone: Expression, Purification, and Determination of Biological Activity in Vitro and in Vivo

Carp growth hormone (cGH) cDNA (Koren et al., 1989) was cloned under the control of λ-phage P_LO_L promoter and λcll ribosomal binding site into pBR322 plasmid to enable its expression in Escherichia coli A1645 that produces constitutively the thermolabile λ repressor c1857. Temperature shift to 42° abolished the repression, resulting in a high level of cGH expression. The bacterially expressed cGH protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on Q-Sepharose column by stepwise elution with NaCl. The bioactive fraction was eluted at 0.2 M NaCl at a yield of 10-15%. This fraction contained predominantly (95%) 21.5-kDa monomeric cGH. The activity of cGH in vitro was bioassayed using Nb2-11C lymphoma cells (containing lactogenic receptors) and 3T3-F442A preadipocyte cells (containing somatogenic receptors). Bioactivity was found to be 0.01 and 6-10% that of human GH, respectively. In vivo cGH activity was measured by weekly ip injection in juvenille carp fed a low (23%) protein diet. Over a 6-week period, cGH increased the growth rate by 38% compared to fish injected with vehicle only. Identical injections with bovine GH yielded only a 21% increase.