title: Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor.
abstract: Our goal is to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its receptor, the parathyroid hormone receptor type-1 (PTHR1). In order to study this interaction, we are aiming to obtain a three-dimensional structure of the PTH-PTHR1 bimolecular complex. Due to the very low expression levels of endogenous PTHR1, a recombinant form is required for structural analysis. However, the extreme hydrophobicity of the transmembrane regions of PTHR1 makes heterologous expression of PTHR1 difficult. Therefore, we sought to express the N-terminal extracellular domain (N-ECD) of PTHR1, a region that plays a pivotal role in ligand interaction. We expressed the N-ECD in both bacterial (Escherichia coli) and insect (Sf9) cells. The form produced in E. coli, a fusion-protein with thioredoxin, is soluble. However, removal of the fusion partner from a partially purified preparation results in dramatic loss of yield of the N-ECD. Expression in Sf9 cells, however, facilitates purification of a soluble form of the N-ECD. Isothermal calorimetry demonstrates that this N-ECD binds PTH-(1-34), albeit with lower affinity than the full-length receptor. This report describes the expression and purification of milligram quantities of the isolated N-ECD of PTHR1. The receptor fragment retains the ability to bind its cognate peptide ligand, an important pre-requisite for subsequent structural studies.
authors: Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
authors: Paul
authors: P
authors: Monaghan
authors: Iwona
authors: I
authors: Woznica
authors: Beenu
authors: B
authors: Moza
authors: Eric J
authors: EJ
authors: Sundberg
authors: Michael
authors: M
authors: Rosenblatt
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007313:Insecta / Q000166:cytology; D017434:Protein Structure, Tertiary / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins