7.5
Protocol 7.1: Cell-Free Expression of Membrane Proteins in the Presence
of Non-ionic Amphipols
7.5.1
Introduction
Obtaining sufficient quantities of functional MPs is often a major bottleneck that hinders structural and
functional studies. A solution to this problem is to perform protein expression in an acellular system
(CFE), which eliminates, at least in part, the limitations of in vivo expression systems resulting from
toxicity or imperfect folding and membrane insertion (Zubay 1973; Rues et al. 2016). Among the many
attractive features of CFE is the possibility it affords to label MPs using limited amounts of isotopically
labeled or unnatural amino acids (Kigawa et al. 1999; LaGuerre et al. 2015). Whereas charged APols
such as A8-35, SAPols, PMAL-B-100, or SMA inhibit the CFE of MPs (Park et al. 2011; Periasamy
et al. 2013), glucosylated non-ionic APols (NAPols) are compatible with it (Bazzacco et al. 2012)
(Figs. 7.8 and 7.9). BR expressed in vitro in the presence of NAPols is properly folded and stays stable
over several months, whereas in DDM it tends to rapidly precipitate (Park et al. 2011; Bazzacco et al.
2012). Because APols tend to be much milder than detergents (see Chap. 5, § 5.5, and Kleinschmidt
and Popot 2014), developing APol-assisted CFE of MPs appears as an attractive alternative to the use
of classical detergents. CFE is carried out using commercial systems or a homemade lysate (Proverbio
et al. 2014). The protocol described below was developed using E.coli lysate, but it should be
extendable to eukaryotic ones.
Comments are in italics and preceded by a pointing hand ( ).
7.5.2
Protocol
• Plasmids for CFE
The RTS pIVEX E. coli vectors are designed for cell-free expression of proteins His 6 -tagged
either at the N- or C-terminus and contain all sequences needed for T7 RNA polymerase-mediated
expression.
• CFE Small-Scale Reaction
Prior to performing a large-scale production of MP, it is essential to optimize the concentration
of NAPol to be used. Small-scale syntheses are therefore carried out in the presence of a range of
NAPol concentrations.
1. Prepare a stock solution of NAPols at 100 g‧L
-1 or 10% w/w. Weigh the powder with an
analytical balance in an Eppendorf tube or, if possible, in a small glass vial, and add water
purified on a Milli-Q Advantage A10 system in order to reach the final concentration.
2. Homogenize the solution with a vortex or by magnetic stirring for at least a couple of hours
before use to reach full rehydration of the lyophilized powder. The solution is then kept at
4
C or frozen at À20
C.
3. Small-scale syntheses are carried out in the presence of 0.5 μg of plasmid and 3, 5, 8, or 10 g‧L
-1
NAPols in 25 or 50 μL of lysate. These quantities of NAPol are larger than is actually necessary
because it is difficult to foresee the amount of expressed proteins. Incubate for 6 h in a
ThermoMixer (Eppendorf) at 700 rpm and 25
C.
7.5 Protocol 7.1: Cell-Free Expression of Membrane Proteins in the Presence of. . .
375
Protocol 7.1: Cell-Free Expression of Membrane Proteins in the Presence
of Non-ionic Amphipols
7.5.1
Introduction
Obtaining sufficient quantities of functional MPs is often a major bottleneck that hinders structural and
functional studies. A solution to this problem is to perform protein expression in an acellular system
(CFE), which eliminates, at least in part, the limitations of in vivo expression systems resulting from
toxicity or imperfect folding and membrane insertion (Zubay 1973; Rues et al. 2016). Among the many
attractive features of CFE is the possibility it affords to label MPs using limited amounts of isotopically
labeled or unnatural amino acids (Kigawa et al. 1999; LaGuerre et al. 2015). Whereas charged APols
such as A8-35, SAPols, PMAL-B-100, or SMA inhibit the CFE of MPs (Park et al. 2011; Periasamy
et al. 2013), glucosylated non-ionic APols (NAPols) are compatible with it (Bazzacco et al. 2012)
(Figs. 7.8 and 7.9). BR expressed in vitro in the presence of NAPols is properly folded and stays stable
over several months, whereas in DDM it tends to rapidly precipitate (Park et al. 2011; Bazzacco et al.
2012). Because APols tend to be much milder than detergents (see Chap. 5, § 5.5, and Kleinschmidt
and Popot 2014), developing APol-assisted CFE of MPs appears as an attractive alternative to the use
of classical detergents. CFE is carried out using commercial systems or a homemade lysate (Proverbio
et al. 2014). The protocol described below was developed using E.coli lysate, but it should be
extendable to eukaryotic ones.
Comments are in italics and preceded by a pointing hand ( ).
7.5.2
Protocol
• Plasmids for CFE
The RTS pIVEX E. coli vectors are designed for cell-free expression of proteins His 6 -tagged
either at the N- or C-terminus and contain all sequences needed for T7 RNA polymerase-mediated
expression.
• CFE Small-Scale Reaction
Prior to performing a large-scale production of MP, it is essential to optimize the concentration
of NAPol to be used. Small-scale syntheses are therefore carried out in the presence of a range of
NAPol concentrations.
1. Prepare a stock solution of NAPols at 100 g‧L
-1 or 10% w/w. Weigh the powder with an
analytical balance in an Eppendorf tube or, if possible, in a small glass vial, and add water
purified on a Milli-Q Advantage A10 system in order to reach the final concentration.
2. Homogenize the solution with a vortex or by magnetic stirring for at least a couple of hours
before use to reach full rehydration of the lyophilized powder. The solution is then kept at
4
C or frozen at À20
C.
3. Small-scale syntheses are carried out in the presence of 0.5 μg of plasmid and 3, 5, 8, or 10 g‧L
-1
NAPols in 25 or 50 μL of lysate. These quantities of NAPol are larger than is actually necessary
because it is difficult to foresee the amount of expressed proteins. Incubate for 6 h in a
ThermoMixer (Eppendorf) at 700 rpm and 25
C.
7.5 Protocol 7.1: Cell-Free Expression of Membrane Proteins in the Presence of. . .
375