INTRACELLULAR IMMUNIZATION OF PROKARYOTIC CELLS AGAINST A BACTERIOTOXIN

Intracellularly expressed antibodies hare been designed to bind and in activate target molecules inside eukaryotic cells, Here we report that an antibody fragment can be used to probe the periplasmic localizatio n of the colicin A N-terminal domain, Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli, To I each their ta rget, they bind to a receptor located on the outer membrane and then a re translocated through the envelope, The N-terminal domain of colicin s is involved in the translocation step and therefore is thought tct i nteract with proteins of tile translocation system, To compete with th is system, a single-chain variable fragment (scFv) directed against th e N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E coli, The periplasmic scFv inhibited the l ethal activity of colicin A and had no effect on the lethal activity o f other colicins, Moreover, the scFv was able to specifically inactiva te hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding, Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with impo rt machinery, This indicates that the N-terminal domain of the toxin i s accessible in the periplasm, Moreover, we show that production of an tibody fragments to interfere with a biological function can be applie d to prokaryotic systems.