Biology Reference
In-Depth Information
Chapter 19
Modifi cations of Wheat Germ Cell-Free System
for Functional Proteomics of Plant Membrane Proteins
Akira Nozawa and Yuzuru Tozawa
Abstract
Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive
architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane
proteins is the diffi culty in producing suffi cient quantities of functional protein for analysis. Here, we des-
cribe three methods for membrane protein production utilizing a wheat germ cell-free protein expression
system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modifi ed with
components necessary to produce functional protein. In this way we have developed modifi cations to a
wheat germ cell-free system for the production of functional membrane proteins. Supplementation of
liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, sup-
plementation of myristic acid enables synthesis of
N
-myristylated peripheral membrane proteins. These
modifi ed cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide
variety of membrane proteins.
Key words
Cell-free protein expression system, Detergents, Liposomes, Membrane proteins,
N
-Myristoylation, Wheat germ
Abbreviations
AtDTC
Arabidopsis thaliana
dicarboxylate/tricarboxylate carrier
bR Bacteriorhodopsin
CBB Coomassie brilliant blue
CF Cell-free
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
FC Fos-choline
MP Membrane protein
RNase Ribonuclease
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
WG
Wheat germ
WGCF
Wheat germ cell-free
