Preparation and comparison of biological properties of recombinant carp (Cyprinus carpio) growth hormone and its Cys-123 to Ala mutant

Abstract

Carp growth hormone (cGH) cDNA, in which Cys-123 was mutated to Ala, was prepared, transferred to the expression vector, expressed in Escherichia coli and the mutant was purified to homogeneity. The mutation only slightly improved yield of the monomeric fraction, indicating that Cys-123 is not involved in improper refolding. As compared to cGH, the mutant (cGH-C123A) exhibited lower binding affinity toward homologous liver receptors and lower bioactivity in a 3T3-F442A preadipocyte bioassay despite the fact that both hormones exhibited almost identical cross-reactivity with anti-cGH antibodies. These results, along with those of a structural comparison to hGH, suggest that Cys-123 is located in the hydrophobic core of the hormone, and is most likely affecting the conformation of the binding site. Dimeric forms of the hormone and its mutant were less active than their respective monomers. Homologous binding experiments using a carp liver microsomal fraction revealed a single receptor population with Kd = 0.77 nM and Bmax = 241 fmol/mg microsomal protein.

Identification of serum GH-binding proteins in the goldfish (Carassius auratus) and comparison with mammalian GH-binding proteins

“…The rcGH used in the present study contains an extra cysteine residue at position 123, as found in the common carp (Fine et al 1993, Law et al 1996. This cysteine does not appear to be directly involved in binding of rcGH to the receptor (Fine et al 1993). Goldfish also possess cDNAs for two GHs; one has cysteine at position 123, whereas this position in the other GH contains a serine (Mahmoud et al 1996).…”

“…The rcGH used in the present study contains an extra cysteine residue at position 123, as found in the common carp (Fine et al 1993, Law et al 1996. This cysteine does not appear to be directly involved in binding of rcGH to the receptor (Fine et al 1993).…”

“…The rcGH used in the present study was the form containing five cysteine residues (Fine et al 1993). Bovine GH (bGH) and rat prolactin (rPRL) were gifts from the National Hormone and Pituitary Program (NIADDK, Baltimore, MD, USA).…”

Preparation and Characterization of Recombinant Dolphin Fish (Coryphaena hippurus) Growth Hormone

“…This latter method does not provide information on whether the purified hormone exists as a monomer or as a mixture of monomers, noncovalent dimers, or oligomers that cannot be identified by SDS-PAGE (11). In contrast, the reports from our laboratory regarding the preparation of three, to date prepared, fish GHs (12)(13)(14)(15) or other proteins (11,16 -22) are always accompanied by a determination of monomer content using a sensitive gel-filtration method. In many cases (12,13,16 -20), we have shown that only the monomers are biologically active despite the other forms' apparent purity on an SDS-polyacrylamide gel or their ability to interact with antibodies.…”

“…Another problem that may hamper the application of GH therapy is a lack of homologous hormones. Although heterologous GHs or related hormones, such as ruminant placental lactogen, have been found to be active in vivo (13,15,(25)(26)(27)(28)(29)(30)(31), their prolonged use may be problematic, as it may elicit an undesired immunological response. Therefore, preparation of homologous GH seems to be quite important.…”