Cloning and expression of cDNA for salmon growth hormone in Escherichia coli

Abstract

ABSTRACTcDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.Human growth hormone now can be produced by genetically engineered organisms and can be used as a therapeutic agent. Salmon growth hormone (sGH) can be synthesized by use of similar techniques, and the massive supply of sGH may be extremely important to fish culture. Growth hormone (GH), together with prolactin and chorionic somatomammotropin (placental lactogen), forms a set of proteins that are structurally related and have partially overlapping biological activities (1). Primary structure analysis of the peptides and of the genes suggests that these hormone genes evolved from a common ancestral origin (1-5). Therefore, these genes provide an excellent model system for studying structurefunction relationships, evolution, and regulation of expression. GH genes have been isolated from several mammalian species and characterized in detail (3,(6)(7)(8). To obtain information about the evolution and the mechanisms of organization of this set of genes, it is essential to compare the structures of these hormone genes isolated from many organisms at various evolutionary stages. No information, however, has been available about lower vertebrates such as fish.

Estradiol Stimulates Growth Hormone Production in Female Goldfish

“…The fusion protein accounted for about 25% of the total cellular proteins (data not shown). In a manner similar to that of other recombinant growth hormones produced in bacteria (Langley et al, 1987;Sekine et al, 1985), GST-gcGH aggregated in the form of an inclusion body in E. coli cells. The GST-gcGH inclusion body had to be refolded before thrombin cleavage to release the gcGH fragment (data not shown).…”

The purification of eukaryotic polypeptides synthesized in Escherichia coli

“…Exposure to alkaline pH (> 9.0) without the use of urea or guanidinium chloride has been used to unfold proteins, renaturation being effected by reduction of pH . However, dilution from urea into buffers at alkaline pH was used to produce active salmon growth hormone (Sekine et al, 1985) and prochymosin . In many of the examples quoted by Olsen (1985), alkaline pH was used at some stage of the refolding processes.…”

“…Less than 2% of the refolded growth hormone preparations were in an aggregated form. Thiol reagents were omitted entirely in unfolding and refolding salmon growth hormone (Sekine et al, 1985). Thus these growth hormones appear to form native disulphide bonds in the absence of exogenous thiol reagents.…”

“…Examples include immunoglobulins (Boss et al, 1984;Cabilly et al, 1984), IL-2 (Liang et al, 1985;Malkovsky et al, 1985), bovine, chicken and salmon growth hormones (George et al, 1985;Gill et al, 1985;Sekine et al, 1985), prochymosin (Green et al, 1985) and urokinase (Winkler et al, 1985). However, with few exceptions (Boss et al, 1984;Cabilly et al, 1984;Marston et al, 1984), quantification of the efficiency with which the proteins are refolded is not provided.…”

Changes in the levels of mRNAs for GH/prolactin/somatolactin family and Pit-1/GHF-1 in the pituitaries of pre-spawning chum salmon

“…GH ssDNA Plasmid DNA containing GH-I cDNA insert (a gift from Kyowa Hakko Co. Ltd, Tokyo, Japan; Sekine et al 1985) was digested by restriction enzyme SacI at one site, and was used as the template for PCR. Synthetic oligonucleotide corresponding to the chum salmon GH-I cDNA (89-108) was used as the primer for PCR.…”