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318 → Ile (11) was expressed from plasmid pPMR132. Cells were lysed after incubation with lysozyme and treated with ribonuclease A and deoxyribonuclease I, and the membranes were isolated by centrifugatbn and suspended in buffer containing the nonionic detergent octyl-poly-oxyethytene (4%). Solubilized material was bound to Ni-Sepharose beads, washed with buffer containing 1% CHAPS, and eluted with imidazote in the same buffer. The pIV-containing fractions were pooled, concentrated, and chromatographed on Bio-Gel A5M (Bio-Rad, Hercules, CA), also in buffer containing 1% CHAPS. Peak fractions were used for the STEM.
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We thank P. Model for suggestions, interest, encouragement, and criticism of the manuscript; B. Lin for STEM specimen preparation; and J. Wall for helpful discussions about the data analysis. Supported by NSF grant MCB93-16625 (M.R.). The Brookhaven National Laboratory STEM is an NIH Supported Resource Center (NIH grant P41-RR01777), with additional support provided by the U.S. Department of Energy, Office of Health and Environmental Research.
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