Overproduction, isolation and determination of the amino-terminal sequence of the SecY protein, a membrane protein involved in protein export in Escherichia coli

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Abstract
The gene product of secY (prlA) is an integral membrane protein with an essential role in protein export in Escherichia coli. When the protein was overproduced, using a plasmid, it was degraded rapidly in the cell. The lon or the htpR mutation did not slow down this degradation, but low-temperature growth conditions (30.degree. C) did so appreciably. On the other hand, the copy number of the pUC8-based plasmid was higher temperature. Thus, the plasmid was first amplified at 42.degree. C and the protein was then accumulated at 30.degree. C. The SecY protein was isolated in sodium dodecyl sulfate (SDS)-denatured form from the membranes of the overproducing cells, using SDS-SDS two-dimensional gel electrophoresis. Its NH2-terminal sequence confirmed the secY reading frame and the translation initiation site assigned previously. The SecY protein does not undergo NH2-terminal processing except for the removal of the initiator methionine.