High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors.
G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.
Bosse-Doenecke
Eva
E
Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany. eva.bosse-doenecke@biochemtech.uni-halle.de
Weininger
Ulrich
U
Gopalswamy
Mohanraj
M
Balbach
Jochen
J
Knudsen
Sanne Møller
SM
Rudolph
Rainer
R
D001483:Base Sequence; D000077270:Exenatide; D052216:Glucagon-Like Peptide 1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D025842:SUMO-1 Protein / Q000378:metabolism; D014688:Venoms / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010455:Peptides; D044168:Receptor, Parathyroid Hormone, Type 1; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014688:Venoms; D052216:Glucagon-Like Peptide 1; D000077270:Exenatide; C051322:glucagon-like peptide 1 (7-37)