TY - JOUR
T1 - Recombinant Carp (Cyprinus carpio) Growth Hormone
T2 - Expression, Purification, and Determination of Biological Activity in Vitro and in Vivo
AU - Fine, Mira
AU - Sakal, Edna
AU - Vashdi, Dorit
AU - Daniel, Violet
AU - Levanon, Avigdor
AU - Lipshitz, Orli
AU - Gertler, Arieh
PY - 1993/1
Y1 - 1993/1
N2 - Carp growth hormone (cGH) cDNA (Koren et al., 1989) was cloned under the control of λ-phage PLOL 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.
AB - Carp growth hormone (cGH) cDNA (Koren et al., 1989) was cloned under the control of λ-phage PLOL 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.
UR - http://www.scopus.com/inward/record.url?scp=0027441235&partnerID=8YFLogxK
U2 - 10.1006/gcen.1993.1008
DO - 10.1006/gcen.1993.1008
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C2 - 8428649
AN - SCOPUS:0027441235
SN - 0016-6480
VL - 89
SP - 51
EP - 61
JO - General and Comparative Endocrinology
JF - General and Comparative Endocrinology
IS - 1
ER -