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 lambda-phage PLOL promoter and lambda cll ribosomal binding site into pBR322 plasmid to enable its expression in Escherichia coli A1645 that produces constitutively the thermolabile lambda repressor c1857. Temperature shift to 42 degrees 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 juvenile 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.
Fine
M
M
Department of Biochemistry and Human Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Sakal
E
E
Vashdi
D
D
Daniel
V
V
Levanon
A
A
Lipshitz
O
O
Gertler
A
A
D000273:Adipose Tissue / Q000166:cytology / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002347:Carps; D002454:Cell Differentiation / Q000187:drug effects; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D013006:Growth Hormone / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012965:Sodium Chloride; D014508:Urea
D011994:Recombinant Proteins; D012965:Sodium Chloride; D014508:Urea; D013006:Growth Hormone; D004247:DNA