The first attempts, carried out with A8-35 and SAPols in an E. coli lysate, gave disappointing
results (Study 7.1 in Table 7.1). Indeed, the two APols, used at 2 gÁL
-1
, were found to inhibit more or
less completely the expression of three test MPs: BR, which comprises seven TM α-helices; the TM
domain of the leader peptidase from E. coli (tLep), a two-TM helix preprotein; and the large-pore
mechanosensitive channel MscL from E. coli, a pentameric protein whose protomers also comprise
two TM α-helices each (Park et al. 2011). The synthesis of BR was found to be completely inhibited
(Fig. 7.5A). The level of inhibition was variable for MscL (Fig. 7.5B) and tLep, but conditions
producing acceptable yields in a reproducible manner could not be identified. A number of parameters
were modified, using a homemade S30 lysate derived from the E. coli Rosetta (lDE3) strain, following
0
10
20
30
40
50
60
70
80
90
100
in vivo, no IB
in vivo, IB
CFE EC
CFE WG
Total
A
Year of first publication
Number of
proteins
4
4
6
2
13
2
22
5
21
3
3
1
6
2
B
α mono
α oligo
β mono
β oligo
in vivo, no IBs
in vivo, IBs
EC-CFE
WG-CFE
Total
Fig. 7.4 Origin of membrane proteins that had been successfully (re)folded in vitro by the end of 2013.
Four types of origins are distinguished: (i) natural expression or overexpression in vivo, barring inclusion
bodies (IBs); (ii) in vivo expression in IBs followed by in vitro folding; (iii) in vitro, cell-free expression in
E. coli lysate (EC-CFE); and (iv) in vitro, cell-free expression in wheat germ lysate (WG-CFE). In A, the
cumulative number of MPs obtained by each method that have been successfully (re)folded is plotted as a
function of time, each protein being entered once for each curve, in the year of the first successful report.
Because some MPs have been (re)folded by different methods and they are counted only once in the curve
summing all production methods, the latter shows fewer proteins than the sum of the individual curves.
Note the rapid increase in the number of folded MPs produced by CFE after 2003. In B, a histogram is
shown of the total number of MPs of each type that have been (re)folded after being obtained by each
method. α(β) mono and α(β) oligo refer, respectively, to α-helical (or β-barrel) MPs that are active either as
monomers or as oligomers. Among MPs produced by CFE (blue bars), monomers with an α-helical TM
region are by far the most numerous (27 out of a total of 36) (From Popot 2014, # 2004 Elsevier Inc. All
rights reserved).
366
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins
results (Study 7.1 in Table 7.1). Indeed, the two APols, used at 2 gÁL
-1
, were found to inhibit more or
less completely the expression of three test MPs: BR, which comprises seven TM α-helices; the TM
domain of the leader peptidase from E. coli (tLep), a two-TM helix preprotein; and the large-pore
mechanosensitive channel MscL from E. coli, a pentameric protein whose protomers also comprise
two TM α-helices each (Park et al. 2011). The synthesis of BR was found to be completely inhibited
(Fig. 7.5A). The level of inhibition was variable for MscL (Fig. 7.5B) and tLep, but conditions
producing acceptable yields in a reproducible manner could not be identified. A number of parameters
were modified, using a homemade S30 lysate derived from the E. coli Rosetta (lDE3) strain, following
0
10
20
30
40
50
60
70
80
90
100
in vivo, no IB
in vivo, IB
CFE EC
CFE WG
Total
A
Year of first publication
Number of
proteins
4
4
6
2
13
2
22
5
21
3
3
1
6
2
B
α mono
α oligo
β mono
β oligo
in vivo, no IBs
in vivo, IBs
EC-CFE
WG-CFE
Total
Fig. 7.4 Origin of membrane proteins that had been successfully (re)folded in vitro by the end of 2013.
Four types of origins are distinguished: (i) natural expression or overexpression in vivo, barring inclusion
bodies (IBs); (ii) in vivo expression in IBs followed by in vitro folding; (iii) in vitro, cell-free expression in
E. coli lysate (EC-CFE); and (iv) in vitro, cell-free expression in wheat germ lysate (WG-CFE). In A, the
cumulative number of MPs obtained by each method that have been successfully (re)folded is plotted as a
function of time, each protein being entered once for each curve, in the year of the first successful report.
Because some MPs have been (re)folded by different methods and they are counted only once in the curve
summing all production methods, the latter shows fewer proteins than the sum of the individual curves.
Note the rapid increase in the number of folded MPs produced by CFE after 2003. In B, a histogram is
shown of the total number of MPs of each type that have been (re)folded after being obtained by each
method. α(β) mono and α(β) oligo refer, respectively, to α-helical (or β-barrel) MPs that are active either as
monomers or as oligomers. Among MPs produced by CFE (blue bars), monomers with an α-helical TM
region are by far the most numerous (27 out of a total of 36) (From Popot 2014, # 2004 Elsevier Inc. All
rights reserved).
366
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins