W0069

Structural Studies of the Unique Replication Protein repA from E. coli Plasmid pKL1. Robert Nicholson, Stanislav Stuchlik^*, William Kay^* and Barry Phipps, Department of Biological Sciences, University of Calgary, Alberta, Canada T2N 1N4, ^*Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada V8W 2Y2

Clinical isolates of Escherichia coli often contain small cryptic plasmids (SCP). One of these, pKL1, contains a single structural gene encoding the protein repA (1). The common presence of SCPs in clinical strains suggests they play a role in pathogenesis. RepA binds to recognition sequences on the pKL1 plasmid and is believed to be involved in initiation of pKL1 replication as well as regulation of its own expression. RepA has also been shown to dramatically amplify other plasmids, which makes this system a potential tool for expression of biotechnologically interesting products. RepA is unique; at an M_r of 18,000 it is smaller than other known replication initiation proteins (M_r approx. 30,000) and has no sequence homology to these or any other known proteins. RepA was purified from bacterial extracts using polyethylene glycol precipitation and ion exchange chromatography. Crystallization trials were performed using the hanging drop method. Initial conditions were found using sparse matrix screens and were optimized with grid screens. Reproducible diffraction quality crystals of repA have been grown with either polyethylene glycol or ammonium sulphate as the precipitant. A complete X- ray diffraction data set has been collected on a 0.3 mm x 0.3 mm x 0.2 mm crystal to 2.5Å on a Bruker/AXS 2K CCD detector at room temperature. The space was I4, a=b=122.1Å and c=43.2Å. Progress toward obtaining heavy atom derivatives and solving the structure will be reported.

1. Burian, J., et al.. (1997) Plasmid 37, 2-14.

Work supported by the Alberta Heritage Foundation for Medical Research.