Biology Reference
In-Depth Information
1
Introduction
Membrane proteins (MPs) play critical roles in a variety of biological
processes such as substrate transport, signal transduction, energy
production, and cellular adhesion. It has been reported that
approximately 30 % of genes in the plant genomes encode MPs.
Despite their abundance, efforts to study the function and structure
of plant MPs have been hampered by their low natural abundance
and the toxicity of MP overexpression in host cells. In the last
decade, cell-free (CF) protein expression systems have attracted
much attention as an alternative strategy for MP production.
The CF system has several advantages that better suit it for MP
production compared to cell-based methodologies. For instance,
MP expression in CF system is decoupled from cell viability allow-
ing for the production of toxic proteins that do not interfere with
translation. In addition, CF systems are open for modifi cation,
allowing supplementation of cofactors, chaperones, lipids, and
detergents in order to produce functional protein.
In this chapter, three methods for the production of MPs using
a wheat germ (WG) CF (WGCF) system are described (Fig.
1
).
The fi rst method is liposome-supplemented WGCF system, which
is suitable for functional analysis of MPs (Fig.
1a
). We previously
reported the development of WGCF-based MP synthesis for
Arabidopsis phosphoenolpyruvate/phosphate translocators [
1
].
The WGCF system-based method has been further modifi ed for the
synthesis of functional mitochondrial carriers, such as
Arabidopsis
thaliana
dicarboxylate/tricarboxylate carrier (AtDTC) [
2
].
This updated method is described in this chapter.
Another method for WGCF synthesis of integral MPs is
through the use of mixed detergents (Fig.
1b
). Through the use of
detergents, MPs are effectively solubilized, allowing for simplifi ed
purifi cation. Recently, we reported mixed-detergent-supplemented
a
b
c
N
-Met
removal
N
-Methionine
aminopeptidase
Micelle
vesicle
Myristoyl-CoA
N
-Myristoyltransferase
CoA
N
-Myristoylated
protein
Vesicle-associated protein
Proteomicelles
Fig. 1
Modifi ed CF synthesis system for MP production. (
a
) CF synthesis of MPs in the presence of liposomes.
Synthesized MPs are associated with liposomes. (
b
) CF synthesis of MPs in the presence of mixed detergent.
MPs are synthesized in the soluble fraction as proteomicelles. (
c
) CF synthesis of MPs in the presence
of myristic acid. Protein that has a myristoylation motif is co-translationally
N
-myristoylated in the presence of
myristic acid by the function of intrinsic
N
-methionine aminopeptidase and
N
-myristoyltransferase
