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.