Preparation of biologically active monomeric recombinant zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) recombinant growth hormones
- 150 Downloads
Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~ 36-fold lower for zGH and ~ 107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.
KeywordsGrowth hormone Fish Recombinant Zebra fish Rainbow trout
We thank Dr. Mike Waters for the gift of PDC-P1-3B9 cells.
- Niv-Spector L, Gonen-Berger D, Gourdou I, Biener E, Gussakovsky EE, Benomar Y, Ramanujan KV, Taouis M, Herman B, Callebaut I, Djiane J, Gertler A (2005) Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists. Biochem J 391:221–230CrossRefPubMedPubMedCentralGoogle Scholar
- Solomon G, Reicher S, Gussakovsky EE, Jomain JB, Gertler A (2006) Large-scale preparation and in vitro characterization of biologically active human placental (20 and 22K) and pituitary (20K) growth hormones: placental growth hormones have no lactogenic activity in humans. Growth Hormon IGF Res 16:297–307CrossRefGoogle Scholar