Amphipol-Assisted Cell-Free Expression
of Membrane Proteins
7
Summary
Cell-free expression of membrane proteins is a way to circumvent some of the problems
encountered during their in vivo expression, among which is the small volume of
membrane that is generally available for storing overexpressed membrane proteins
and their toxicity when expressed in excessive amounts. Because of their mildness,
amphipols appear as an attractive medium in which to solubilize and allow folding of
membrane proteins expressed in vitro in cell lysates. Relatively few attempts at doing so
have been described to date. They indicate (i) that all ionic polymers tested thus far
interfere with the synthesis of α-helical membrane proteins and (ii) that two non-ionic
polymers, NVoy and a glucosylated non-ionic amphipol, do allow their synthesis,
folding, and solubilization in good yields.
7.1
Introduction
Very few membrane proteins (MPs) are produced naturally in amounts sufficient for structural studies.
As discussed in Chap. 1, this is mostly the consequence of two factors. First, the volume of the plasma
membrane of a cell is small as compared to that of the cytosol. Even if both compartments are packed
full with proteins (typically ~200 gÁL
-1 in the cytosol, generally 1–3 g protein per g of lipid in the
plasma membrane), the ratio in mass of MPs to proteins in general is necessarily small. Second, many
MPs of great physiological importance are involved in signaling, which does not necessarily require
them to be present in very many copies. In a muscle fiber, for instance, the nicotinic acetylcholine
receptor (nAChR) is densely packed under the motor nerve terminals, but these cover only ~0.1% of
the total surface of the fiber. Similarly, most of the G protein-coupled receptors (GPCRs), which
control so many regulatory mechanisms, need not be present in large amounts, because their response
to a stimulus is enormously amplified by downstream events. This explains why the first MPs to be
purified in sizable amounts were typically involved in energy production, such as bacteriorhodopsin
(BR) or photosynthetic reaction centers, or in mass transport of solutes, such as the porins from
bacterial outer membranes. The nicotinic acetylcholine receptor (nAChR) was first purified from the
electric organs of either Electrophorus or Torpedo, where it is diverted from its usual signaling
function and massively expressed so as to produce large electric discharges. In many cases, such as
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_7
361
of Membrane Proteins
7
Summary
Cell-free expression of membrane proteins is a way to circumvent some of the problems
encountered during their in vivo expression, among which is the small volume of
membrane that is generally available for storing overexpressed membrane proteins
and their toxicity when expressed in excessive amounts. Because of their mildness,
amphipols appear as an attractive medium in which to solubilize and allow folding of
membrane proteins expressed in vitro in cell lysates. Relatively few attempts at doing so
have been described to date. They indicate (i) that all ionic polymers tested thus far
interfere with the synthesis of α-helical membrane proteins and (ii) that two non-ionic
polymers, NVoy and a glucosylated non-ionic amphipol, do allow their synthesis,
folding, and solubilization in good yields.
7.1
Introduction
Very few membrane proteins (MPs) are produced naturally in amounts sufficient for structural studies.
As discussed in Chap. 1, this is mostly the consequence of two factors. First, the volume of the plasma
membrane of a cell is small as compared to that of the cytosol. Even if both compartments are packed
full with proteins (typically ~200 gÁL
-1 in the cytosol, generally 1–3 g protein per g of lipid in the
plasma membrane), the ratio in mass of MPs to proteins in general is necessarily small. Second, many
MPs of great physiological importance are involved in signaling, which does not necessarily require
them to be present in very many copies. In a muscle fiber, for instance, the nicotinic acetylcholine
receptor (nAChR) is densely packed under the motor nerve terminals, but these cover only ~0.1% of
the total surface of the fiber. Similarly, most of the G protein-coupled receptors (GPCRs), which
control so many regulatory mechanisms, need not be present in large amounts, because their response
to a stimulus is enormously amplified by downstream events. This explains why the first MPs to be
purified in sizable amounts were typically involved in energy production, such as bacteriorhodopsin
(BR) or photosynthetic reaction centers, or in mass transport of solutes, such as the porins from
bacterial outer membranes. The nicotinic acetylcholine receptor (nAChR) was first purified from the
electric organs of either Electrophorus or Torpedo, where it is diverted from its usual signaling
function and massively expressed so as to produce large electric discharges. In many cases, such as
# Springer International Publishing AG, part of Springer Nature 2018
J. -L. Popot, Membrane Proteins in Aqueous Solutions, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-73148-3_7
361