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. Photoa ffinity labeling with the adrenergic Ligand [I-125]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergen ic region between the fusion partners after production in E. coli. The simp le 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 coup ling to stimulatory GTP-binding protein. The production level with 3 pmol r eceptor fusion protein per mg membrane protein in a crude membrane preparat ion was significantly higher than those reported for other beta(2) adrenerg ic receptor constructs in E. coli. After solubilization with dodecanoyl suc rose, the fusion protein was purified to near homogeneity by affinity chrom atography on immobilized Ni2+ ions (binding to a C-terminal His(6)-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher p roduction levels, the fusion protein preceded by an insect signal peptide w as produced in baculovirus-infected insect cells. As expected, the producti on 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 t he insect cells bound adrenergic ligands and activated heterotrimeric GTP-b inding proteins with biochemical properties comparable to that of the unfus ed receptor. (C) 2000 Elsevier Science B.V. All rights reserved.