Preparation of a highly translocation-competent proOmpA/SecB complex

Abstract

Methods for reproducibly preparing highly translocation‐competent proOmpA were developed. Only a competent form of proOmpA was sorted out from incompetent one using SecB, a translocation‐dedicated chaperone, as a probe. Trypsin digestion revealed that the incompetent form of proOmpA was partially folded at its N‐terminus, consistent with the jamming of proOmpA within translocon. Although the incompetent form of proOmpA was not active as to topology inversion of SecG, the isolated proOmpA/SecB complex had recovered the ability of SecG inversion. These results let us prepare a proOmpA/SecB complex both in vivo and in vitro that is highly translocation‐competent. E. coli cells harboring a plasmid, in which ompA and secB were encoded as a synthetic operon, accumulated the proOmpA/SecB complex in the cytosol. The complex, purified by means of a His tag attached to SecB, was found to be translocation‐competent as revealed by the occurrence of SecG inversion, although the signal peptide of proOmpA was sensitive to proteolytic digestion. ProOmpA, in vitro synthesized by means of a continuous exchange cell free system in the presence of SecB‐His, was purified as a complex with SecB, which was active as to SecG inversion as well.