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Development of a Minimal Cell‐Free Translation System for the Synthesis of Presecretory and Integral Membrane Proteins
Author(s) -
Kuruma Yutetsu,
Nishiyama Kenichi,
Shimizu Yoshihiro,
Müller Matthias,
Ueda Takuya
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049553u
Subject(s) - periplasmic space , membrane protein , integral membrane protein , microbiology and biotechnology , translation (biology) , biology , protein biosynthesis , cell free protein synthesis , biochemistry , chromosomal translocation , membrane , chemistry , escherichia coli , gene , messenger rna
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 (MtlA) 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.