Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein

We are testing a strategy for creating three-dimensional crystals of integr al membrane proteins which involves the addition of a large soluble domain to the membrane protein to provide crystallization contacts. As a test of t his strategy we designed a fusion between the membrane protein bacteriorhod opsin (BR) and the catalytic subunit of aspartyl transcarbamylase from Esch erichia coli. The fusion protein (designated BRAT) was initially expressed in E. coli at 51 mg/liter of culture, to yield active aspartyl transcarbamy lase and an unfolded bacterio-opsin (BO) component, In Halobacterium salina rum, BRAT was expressed at a yield of 7 mg/liter of culture and formed a hi gh-density purple membrane. The visible absorption properties of BRAT were indistinguishable from those of BR, demonstrating that the fusion with aspa rtyl transcarbamylase had no effect on BR structure. Electron microscopy of BRAT membrane sheets showed that the fusion protein was trimeric and organ ized in a two-dimensional crystalline lattice similar to that in the BR pur ple membrane. Following solubilization and size-exclusion purification in s odium dodecyl sulfate, the BO portion of the fusion was quantitatively refo lded in tetradecyl maltoside (TDM). Ultracentrifugation demonstrated that B R and BRAT-TDM mixed micelles had molecular masses of 138 and 162 kDa, resp ectively, with a stoichiometry of one protein per micelle. High TDM concent rations (>20 mM) were required to maintain BRAT solubility, hindering three -dimensional crystallization trials. We have demonstrated that BR can funct ionally accommodate massive C-terminal fusions and that these fusions may b e expressed in quantities required for structural investigation in H. salin arum. (C) 1999 Academic Press.