The long term objective is to understand the mode of action of colicin E1 immunity protein which protects bacteria against colicin killing. Purification of the immunity protein may allow the development of an in vitro assay for its activity. Because of its low abundance, cloning of the immunity protein into high expression vectors has been completed. Over-production of the cloned protein in a consistent manner will be systematically pursued. Specific antibodies that recognize the immunity protein will be further purified from the total immunoglobulin fraction which is immunoblot positive. Additional antibodies against synthetic peptides corresponding to the hydrophilic domains of the immunity protein will be prepared if needed. These antibodies will be used to purify the immunity protein by affinity chromatography in conjunction with more conventional protein purification techniques. Antibodies against the synthetic peptides will be used to probe the membrane topography of the protein by immunofluorescence and electron microscopy in bacterial vesicles. A new fluorescence assay will be used to test the possible activity of the purified immunity protein in reconstituted liposomes against colicin channels. Binding, proteolytic cleavage or chemical modification will be explored as possible modes of channel inactivation. Temperature-sensitive immunity mutants will be screened for after in vitro mutagenesis of the plasmid. Colicin E1 fragments generated by the genetic manipulation of the Col E1 plasmid will be tested for channel activity in liposomes. The molecularities of other E1-type colicins (K, A, Ia and Ib) will be determined with a new stopped-flow ion-flux assay.
| Shanafelt, A B; Goldman, K M; Kastelein, R A et al. (1987) Cloning and overexpression of the colicin E1 immunity gene. Plasmid 17:261-4 |
| Bruggemann, E P; Kayalar, C (1986) Determination of the molecularity of the colicin E1 channel by stopped-flow ion flux kinetics. Proc Natl Acad Sci U S A 83:4273-6 |
| Kayalar, C; Duzgunes, N (1986) Membrane action of colicin E1: detection by the release of carboxyfluorescein and calcein from liposomes. Biochim Biophys Acta 860:51-6 |
| Suit, J L; Fan, M L; Kayalar, C et al. (1985) Genetic study of the functional organization of the colicin E1 molecule. J Bacteriol 161:944-8 |
| Goldman, K; Suit, J L; Kayalar, C (1985) Identification of the plasmid-encoded immunity protein for colicin E1 in the inner membrane of Escherichia coli. FEBS Lett 190:319-23 |
