Bacterial expression of prolactin family proteins.
Complementary DNAs of three recombinant proteins related to the prolactin family: ovine placental lactogen (oPL), ovine prolactin (oPRL), and rabbit soluble extracellular domain of prolactin receptor (rbPRLR-ECD) were subcloned by different methods and inserted into prokaryotic expression plasmids. Escherichia coli cells transformed with those plasmids overexpressed the respective proteins either by induction or constitutively, resulting in accumulation of the recombinant proteins in insoluble inclusion bodies, which were subsequently purified, used for refolding and purifying of the proteins by one-step chromatography. The isolated oPL, oPRL, and rbPRLR-ECD were biologically active over >95% pure monomers. Ten-liter bacterial culture yielded hundreds of milligrams or more than gram quantities of recombinant proteins. The methodology described in the present chapter allows large-scale preparation of pure, monomeric, biologically active oPL, oPRL, and rbPRLR-ECD suitable for performing in vitro and in vivo experiments.
Gertler
Arieh
A
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Israel.
D000818:Animals; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D002479:Inclusion Bodies; D010928:Placental Lactogen / Q000096:biosynthesis* / Q000737:chemistry; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis* / Q000737:chemistry; D011817:Rabbits; D011981:Receptors, Prolactin / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012756:Sheep; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D010928:Placental Lactogen