Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells.
The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.
Hampe
W
W
Abteilung für Molekulare Membranbiologie, Max-Planck-Institut für Biophysik, Frankfurt/M., Germany.
Voss
R H
RH
Haase
W
W
Boege
F
F
Michel
H
H
Reiländer
H
H
D018528:ATP-Binding Cassette Transporters; D000319:Adrenergic beta-Antagonists; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D003978:Diazomethane; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D019920:Photoaffinity Labels; D010869:Pindolol / Q000031:analogs & derivatives; D011943:Receptors, Adrenergic, beta / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D018411:Spodoptera / Q000378:metabolism*; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D000319:Adrenergic beta-Antagonists; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D019920:Photoaffinity Labels; D011943:Receptors, Adrenergic, beta; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C047975:cyanopindolol; D003978:Diazomethane; D010869:Pindolol; D019204:GTP-Binding Proteins