title: Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins.
abstract: Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.
authors.affiliation: Laboratoire de Biologie Moleculaire et de Génie Génétique, Université de Liège, Sart Tilman, Belgium.
authors.forename: D
authors.initials: D
authors.lastname: Swennen
authors.forename: F
authors.initials: F
authors.lastname: Rentier-Delrue
authors.forename: B
authors.initials: B
authors.lastname: Auperin
authors.forename: P
authors.initials: P
authors.lastname: Prunet
authors.forename: G
authors.initials: G
authors.lastname: Flik
authors.forename: S E
authors.initials: SE
authors.lastname: Wendelaar Bonga
authors.forename: M
authors.initials: M
authors.lastname: Lion
authors.forename: J A
authors.initials: JA
authors.lastname: Martial
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D011388:Prolactin