Bacterial expression and purification of the ovine type II bone morphogenetic protein receptor ectodomain.
Bone morphogenetic proteins (BMPs) are essential for a wide range of developmental processes. They signal through type I and type II serine/threonine kinase receptors, and differ from other TGF-beta family members in that the type II receptor binds with a lower affinity than the type I. Here, we describe the development of various Escherichia coli expression systems for the extracellular domain of the ovine type II bone morphogenetic protein receptor. In order to facilitate disulfide bond formation and protein solubility, BMPRII was expressed fused to bacterial thioredoxin, which, following cleavage, could be purified using several chromatography steps. Although this material migrated as a single band in denaturing PAGE, native-PAGE indicated heterogeneity, and this protein could not be crystallised. When expressed alone, either containing a histidine tag or as an untagged protein, the receptor ectodomain was deposited as insoluble inclusion bodies. Protein subjected to in vitro refolding procedures exhibited multiple species following anion exchange chromatography and size exclusion chromatography, as visualised on native-PAGE. Separation of these species could be achieved using a MonoP chromatofocusing matrix. One of these separated fractions, representing about 5% of the starting material, was amenable to crystallisation, and furthermore exhibited biological activity. Crystals of the histidine-tagged form were shown to diffract weakly, whereas crystals of the native form grew in two different morphologies, and diffracted to a resolution of 1.2A.
Mace
Peter D
PD
Department of Biochemistry, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin 9001, New Zealand. peter.mace@stonebow.otago.ac.nz
Cutfield
John F
JF
Cutfield
Sue M
SM
D001483:Base Sequence; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D017931:DNA Primers; D011994:Recombinant Proteins; D052006:Bone Morphogenetic Protein Receptors, Type II