Solubilization and purification of the human ETB endothelin receptor produced by high-level fermentation in Pichia pastoris.
In the present report, the successful solubilization and purification of the ETB receptor heterologously produced in the methylotrophic yeast P. pastoris is described for the first time. In comparison to the baculovirus system where successful production, solubilization and purification have already been reported, handling and up-scaling of recombinant P. pastoris cells was much easier and less time consuming. Recombinant P. pastoris clones producing two different ETB receptor constructs were grown in a fermenter to a density of about 360 g/l. After induction with methanol, a production level of maximally 45 pmol/mg was obtained, a value which is in the range of that reported for baculovirus-infected insect cells. A method for the large-scale preparation of membranes was established. Solubilization of the recombinant ETB receptor was achieved with the detergent n-dodecyl-/beta-D-maltopyranoside. The stability of the solubilized and ligand-bound receptor was examined in detail. Subsequently, two purification methods for two different receptor constructs were tested and a large-scale procedure for isolation of recombinant receptor was established. In general, the purification methods described herein will be adaptable to other G protein-coupled receptors heterologously produced in heterologous expression systems including P. pastoris.
Schiller
H
H
Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Frankfurt/M., Germany.
Molsberger
E
E
Janssen
P
P
Michel
H
H
Reiländer
H
H
D001665:Binding Sites; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003902:Detergents; D005285:Fermentation; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D008320:Maltose / Q000031:analogs & derivatives*; D010843:Pichia; D010957:Plasmids; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D014170:Transformation, Genetic
D003902:Detergents; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; C411362:dodecyl maltopyranoside; D008320:Maltose