these observations bode well for the use of NAPols for CFE of MPs, difficulties in scaling their
production up to a marketable level have hitherto prevented exploring the generality of the observations made with BR.
7.4
Conclusions and Perspectives
From the data presented in § 7.3, it will have been apparent that the use of APols for cell-free
expression of MPs is only in its infancy: only a handful of studies have been published, some of
which yielded negative results – inhibition of synthesis – and those in which synthesis proceeded
normally have been limited, as of now, to two different polymers and a single structural type of MPs,
featuring seven TM α-helices.
Yet, extremely encouraging results have been obtained with the two non-ionic polymers tested,
NVoy, shown to be compatible with the expression of seven GPCRs of various classes (Klammt et al.
2011), and a non-ionic, glucosylated NAPol, tested on the sole BR (Bazzacco et al. 2012). In both
studies, reasonable yields were achieved (in the 0.4–1 gÁL
-1 range), and most of the MP was recovered
in the form of soluble MP/polymer complexes. For the one GPCR whose percentage of functional
folding was determined, ~10% of the protein was found to bind its ligands and could be purified by
affinity chromatography. For BR, the yield of functional folding was estimated to be at least two-thirds
of the protein present in the IMAC-purified sample (Popot et al. 2011). It would be highly desirable, of
course, to examine more in depth the factors that determine the folding yield, as well as to compare
more systematically the yields obtained when expressing MPs in the presence of the polymer and when
letting them precipitate and recovering them by detergent solubilization followed by transfer to the
polymer (Klammt et al. 2011). It is also necessary to apply these two types of molecules to CFE of a
wider variety of MPs, including oligomeric MPs and MPs that fold into β-barrels. For NVoy, carrying
out these investigations ought to be relatively straightforward, as the molecule is simple to synthesize
and commercially available. The synthesis of glucosylated NAPols is more cumbersome, and extensive studies of their use will have to wait for it to be scaled up and the product marketed, or for easierto-synthesize NAPols to be developed.
It is worth noting that, with both non-ionic polymers, it has been shown that homogeneous or
relatively homogeneous MP/polymer complexes can be obtained and studied by such structural
methods as radiation scattering (BR/NAPol complexes), electron microscopy (GPCR/NVoy ones),
and NMR (both types of complexes) (see Klammt et al. 2011; Bazzacco et al. 2012; Sharma et al.
2012). Furthermore, it can be expected that MPs complexed by non-ionic polymers might be easier to
crystallize than charged ones (cf. Chap. 11), as well as more generally usable for ligand-binding
studies, because of a lower background binding of cationic ligands (cf. Chaps. 5 and 13). The scattered
results available on the stability of MPs trapped with either NVoy (Klammt et al. 2011) or NAPols
(Bazzacco et al. 2012) suggest that the two polymers are particularly mild. MPs trapped in these
polymers may therefore stand a good chance at being amenable to experiments that require them to
remain properly folded for days or weeks, such as extensive NMR measurements or crystallization
attempts. Furthermore, the possibility to express MPs directly in the presence of non-ionic polymers
and, therefore, to avoid any contact with detergents opens up the prospect of gaining access to MPs that
are too fragile to be either solubilized by detergents following precipitation or expressed in their
presence.
As compared to charged APols and, in particular, A8-35, NVoy and NAPols suffer from the
disadvantage that no or very few labeled or tagged derivatives exist yet (Chap. 4). Perdeuteration of
any of these polymers would be difficult to carry out, and, if doable, would certainly be very expensive.
No fluorescent derivatives of either polymer have been synthesized yet, and a single tagged one has
7.4 Conclusions and Perspectives
373
production up to a marketable level have hitherto prevented exploring the generality of the observations made with BR.
7.4
Conclusions and Perspectives
From the data presented in § 7.3, it will have been apparent that the use of APols for cell-free
expression of MPs is only in its infancy: only a handful of studies have been published, some of
which yielded negative results – inhibition of synthesis – and those in which synthesis proceeded
normally have been limited, as of now, to two different polymers and a single structural type of MPs,
featuring seven TM α-helices.
Yet, extremely encouraging results have been obtained with the two non-ionic polymers tested,
NVoy, shown to be compatible with the expression of seven GPCRs of various classes (Klammt et al.
2011), and a non-ionic, glucosylated NAPol, tested on the sole BR (Bazzacco et al. 2012). In both
studies, reasonable yields were achieved (in the 0.4–1 gÁL
-1 range), and most of the MP was recovered
in the form of soluble MP/polymer complexes. For the one GPCR whose percentage of functional
folding was determined, ~10% of the protein was found to bind its ligands and could be purified by
affinity chromatography. For BR, the yield of functional folding was estimated to be at least two-thirds
of the protein present in the IMAC-purified sample (Popot et al. 2011). It would be highly desirable, of
course, to examine more in depth the factors that determine the folding yield, as well as to compare
more systematically the yields obtained when expressing MPs in the presence of the polymer and when
letting them precipitate and recovering them by detergent solubilization followed by transfer to the
polymer (Klammt et al. 2011). It is also necessary to apply these two types of molecules to CFE of a
wider variety of MPs, including oligomeric MPs and MPs that fold into β-barrels. For NVoy, carrying
out these investigations ought to be relatively straightforward, as the molecule is simple to synthesize
and commercially available. The synthesis of glucosylated NAPols is more cumbersome, and extensive studies of their use will have to wait for it to be scaled up and the product marketed, or for easierto-synthesize NAPols to be developed.
It is worth noting that, with both non-ionic polymers, it has been shown that homogeneous or
relatively homogeneous MP/polymer complexes can be obtained and studied by such structural
methods as radiation scattering (BR/NAPol complexes), electron microscopy (GPCR/NVoy ones),
and NMR (both types of complexes) (see Klammt et al. 2011; Bazzacco et al. 2012; Sharma et al.
2012). Furthermore, it can be expected that MPs complexed by non-ionic polymers might be easier to
crystallize than charged ones (cf. Chap. 11), as well as more generally usable for ligand-binding
studies, because of a lower background binding of cationic ligands (cf. Chaps. 5 and 13). The scattered
results available on the stability of MPs trapped with either NVoy (Klammt et al. 2011) or NAPols
(Bazzacco et al. 2012) suggest that the two polymers are particularly mild. MPs trapped in these
polymers may therefore stand a good chance at being amenable to experiments that require them to
remain properly folded for days or weeks, such as extensive NMR measurements or crystallization
attempts. Furthermore, the possibility to express MPs directly in the presence of non-ionic polymers
and, therefore, to avoid any contact with detergents opens up the prospect of gaining access to MPs that
are too fragile to be either solubilized by detergents following precipitation or expressed in their
presence.
As compared to charged APols and, in particular, A8-35, NVoy and NAPols suffer from the
disadvantage that no or very few labeled or tagged derivatives exist yet (Chap. 4). Perdeuteration of
any of these polymers would be difficult to carry out, and, if doable, would certainly be very expensive.
No fluorescent derivatives of either polymer have been synthesized yet, and a single tagged one has
7.4 Conclusions and Perspectives
373