Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

Yutetsu Kuruma, Ken Ichi Nishiyama, Yoshihiro Shimizu, Matthias Müller, Takuya Ueda

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtIA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/ SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.

Original languageEnglish
Pages (from-to)1243-1251
Number of pages9
JournalBiotechnology Progress
Volume21
Issue number4
DOIs
Publication statusPublished - 2005 Jul 1
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology

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