Chemistry Reference
In-Depth Information
protein 2 (UCP2) [
41
]. However insertion of large amounts of expressed protein in
the cell membrane is often not well tolerated by the host [
42
,
43
]. Compromised cell
viability may arise from changes in lipid bilayer fluidity [
44
], or by overwhelming
the cytoplasmic protein translocation machinery, fundamentally altering the com-
position of both the cell envelope and cytoplasmic proteome [
45
-
47
]. Special
strains have been developed that better tolerate the stresses of toxic protein expres-
sion [
48
] that have proven useful for some membrane proteins [
49
,
50
]. However,
even in these systems, many membrane proteins are produced at levels that are too
low to facilitate structural studies by solution NMR.
One strategy that has been used to circumvent these toxicity issues is to express the
membrane protein as inclusion bodies, thereby avoiding insertion into the membrane
[
51
-
53
]. These insoluble aggregates of misfolded proteins are usually non-toxic to
the host cell [
52
]. Since they are also typically resistant to proteolytic cleavage,
expression levels as high as 25% of the total cell protein has been attained through
this approach [
53
]. While overexpression of some membrane proteins spontaneously
gives rise to inclusion body formation, most notably with
-barrels missing their
signal sequences, and mitochondrial carrier proteins [
54
,
55
], for other proteins it is
possible to use fusion tags that target them to inclusion bodies [
56
,
57
]. For example,
expression of a trp operon L gene that has been modified to allow translation through
its native stop codon generates a polypeptide of 105 residues called trp
b
LE [
58
,
59
].
This polypeptide has a strong tendency to form inclusion bodies either when
expressed on its own or when expressed as an N-terminal fusion to smaller membrane
proteins (i.e., one to two transmembrane (TM) helices [
60
-
65
]).
Many solution NMR structures of polypeptides comprised of a single TM helix
have been produced by inclusion body targeting [
62
,
63
,
65
-
67
]. In the case of
larger proteins that span the membrane multiple times, the development of high-
yielding refolding protocols can present a significant impediment. Nonetheless this
strategy has proven to be particularly useful for the production of
D
b
-barrel
structures, e.g., the bacterial palmitoyltransferase PagP [
68
], outer membrane
proteins OmpA [
69
,
70
] and OmpX [
71
], the pH-sensitive OmpG porin [
72
], and
the mitochondrial voltage-dependent anion channel VDAC-1 [
73
]. In addition,
there are a number of examples of polytopic helical membrane proteins that have
been expressed and refolded from inclusion bodies, e.g., a mammalian G-protein
coupled receptor (GPCR) [
29
,
56
,
74
], the Y-2 receptor [
75
] bacteriorhodopsin
[
76
], and a range of mitochondrial carriers [
55
,
77
,
78
]. These examples suggest
some potential for inclusion body targeted expression of larger proteins, although
no membrane protein NMR structure has yet been produced by this approach for
any protein containing more than two TM segments.
2.3 Cell-Free Expression Approaches
A promising approach that circumvents complications arising from membrane
protein toxicity or refolding is the cell free expression system comprised of purified
