title: Production and purification of recombinant somatolactin beta and its effects on melanosome aggregation in zebrafish.
abstract: A second form of somatolactin, somatolactin beta (SLbeta), was recently discovered in zebrafish (Danio rerio). This novel subtype of somatolactin is distantly related to somatolactin alpha (SLalpha) found in teleost species and is produced in a different region of the pituitary. To date, no physiological study of SLbeta has been reported. In order to study the physiological functions of SLbeta, recombinant SLbeta protein has been produced and purified. The cDNA of zebrafish SLbeta was cloned into a pET100 bacteria expression vector and His-tagged fusion proteins were produced in BL21 (DE3) Escherichia coli cells. The majority of recombinant somatolactins produced by E. coli were isolated in inclusion bodies although a small percentage of recombinant proteins (<1%) were also found in soluble form. Fusion proteins were solubilized from inclusion bodies using 6M guanidine hydrochloride. Pure recombinant somatolactins were obtained by affinity purification. The estimated molecular weight of 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis correlates with the molecular mass calculated from the deduced amino acid sequence of SLbeta. Thereafter, specific polyclonal antibodies against the recombinant SLbeta were developed. These antibodies recognized specifically a group of cells located in the anterior pars intermedia of the pituitary. The antibodies did not react with SLalpha, growth hormone or prolactin cells in the zebrafish pituitary glands. Furthermore, recombinant SLbeta induced melanosome aggregation in a concentration-dependent manner in skin of zebrafish scales. Significant melanosome aggregation was observed in zebrafish melanophores at a concentration of 1 microg/ml. These results, combined with previous reports demonstrate that the recombinant SLbeta proteins produced here are bioactive. The function of inducing melanosome aggregation is conserved among the somatolactin functions.
authors.affiliation: Department of Biology, Howell Science Complex, East Carolina University, 1000 E. 5th Street, Greenville, NC 27858, USA.
authors.forename: Nhu
authors.initials: N
authors.lastname: Nguyen
authors.forename: Masazumi
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D029941:Fish Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008544:Melanocytes / Q000502:physiology / Q000648:ultrastructure; D020460:Melanosomes / Q000187:drug effects* / Q000502:physiology; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D010907:Pituitary Hormones / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D012867:Skin / Q000187:drug effects / Q000648:ultrastructure; D015027:Zebrafish / Q000033:anatomy & histology / Q000502:physiology*; D029961:Zebrafish Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D006023:Glycoproteins; D010907:Pituitary Hormones; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C064448:somatolactin protein, fish