OVEREXPRESSION, REFOLDING, AND CRYSTALLIZATION OF AN 80 KD OUTER MEMBRANE PROTEIN. Susan Buchanan¹, Barbara Smith¹, Lalitha Venkatramani², Dick van der Helm², and Johann Deisenhofer^1, Howard Hughes Medical Institute, UT Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75235-9050^1, Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019²

Ferric enterobactin receptor (FepA), an outer membrane protein from Esherichia coli, has been overexpressed to produce large quantides of insoluble cytoplasmic inclusion bodies. The inclusion bodies have been solubilized in urea and refolded using a combination of sulfobetaine 3-14 and sodium dodecylsulfate. The refolded protein was subsequently purified by FPLC using anion exchange and gel filtration chromatography. Refolded FepA was crystallized according to methods developed for native (membrane-inserted) FepA; the resulting crystals have the identical space group and unit cell dimensions determined for native FepA crystals. A low temperature native data set has been collected to 2.9 Å resolution and a search for heavy atom derivatives is in progress, using crystals from both native and refolded sources. Current yields from the inclusion body expression system are approximately 10 mg/1, making this method suitable for structual studies of other outer membrane proteins.