RAPID PURIFICATION OF NATIVE SECA FROM ESCHERICHIA-COLI - DEVELOPMENTOF A NEW AFFINITY-CHROMATOGRAPHY PROCEDURE

The SecA protein occupies a pivotal position in the public protein exp ort pathway in Escherichia coli. The multifunctional SecA protein reco gnizes cytoplasmic factors associated with export including the presec retory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of Sec A to bind ATP was the basis for the development of a novel, rapid puri fication scheme involving a single chromatographic step. Affinity chro matography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, a nd active protein was purified to greater than 90% homogeneity. The pr otein isolated by this procedure retained the previously described ATP ase and RNA-binding activities of SecA. This approach should permit th e rapid purification of SecA homologs from a variety microorganisms.