Bacterial production and purification of the fish pituitary hormone somatolactin.
Somatolactin, a pituitary hormone belonging to the growth hormone/prolactin family, is produced in the intermediate lobe of teleost pituitary. To date, the functions of this new hormone and the target tissues are unknown. A Solea senegalensis somatolactin (ssSL) cDNA has previously been cloned and isolated. Here we have inserted this cDNA into a pET-3a plasmid in order to produce recombinant ssSL in E. coli BL21 (DE3) cells. The protein induced was isolated from inclusion bodies by a solubilization-renaturation procedure originally developed to generate native disulfide bonds, to get putative active proteins. The recombinant somatolactin was further purified to homogeneity by gel filtration on FPLC. The estimated molecular weight of 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis agrees well with the molecular mass calculated from the translated cDNA sequence and with native somatolactin (SL). The recombinant protein showed electrophoretic mobility identical to that of one of the native forms of SL secreted in vitro by cultured pituitaries from sole. Another native SL expressed in S. senegalensis represented a glycosylated modified hormone as shown by N-glycosidase treatment. Further, recombinant SL was recognized by an anti-native SL antibody and used to generate polyclonal sera reactive with the native pituitary hormone. To date, this represents the first recombinant SL protein isolated in sufficient quantities for biophysical and biochemical investigation and for studies on its physiological actions.
Pendón
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Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, Puerto Real, Spain.
Martínez-Barberá
J P
JP
Ortíz
M
M
Valdivia
M M
MM
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029941:Fish Proteins; D005399:Fishes / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D002479:Inclusion Bodies / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry; D010907:Pituitary Hormones / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D029941:Fish Proteins; D006023:Glycoproteins; D010907:Pituitary Hormones; D011994:Recombinant Proteins; C064448:somatolactin protein, fish; D008623:Mercaptoethanol; D006026:Glycoside Hydrolases