Recombinant expression and purification of the N-terminal extracellular domain of the parathyroid hormone receptor.
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.
Monaghan
Paul
P
Department of Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Woznica
Iwona
I
Moza
Beenu
B
Sundberg
Eric J
EJ
Rosenblatt
Michael
M
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*
D044168:Receptor, Parathyroid Hormone, Type 1; D011994:Recombinant Proteins