Independent of the method to store a protein, it is necessary to
assess the quality of the sample after storage, i.e., before it is used in
downstream applications. This can be done using a batch-to-batch
consistency protocol combining the basic QC tests described above
(Subheadings 1.1–1.3). It is essential that the protein’s physicochemical properties are not altered during storage. Processes such
as oxidation, precipitation, or (partial) degradation may impede the
study of protein structure and function or render it completely
impossible. This problem has been extensively studied for proteins
that are used as therapeutics, and it has been found that the most
crucial aspect in stabilizing the protein, and allowing storage for
18–24 months without loss of integrity and functionality, is the
final buffer composition; thus, it is worth investing time and consumables in the optimization of the buffer used for storage, which
may be distinct from that used downstream.
1.4.5 Membrane Protein
Buffer Optimization
Membrane proteins have large surface patches of hydrophobic
residues required for their insertion into or interaction with membranes. In aqueous solution, these patches promote protein aggregation through strong hydrophobic interactions. Thus, the
conditions for solubilizing membrane proteins are distinct from
those of cytoplasmic proteins. The most common method for
solubilizing (integral) membrane proteins is the addition of detergents or amphipols—polymers with a hydrophilic backbone decorated with hydrophobic sidechains, making them amphiphilic and
thus enabling them to stabilize membrane proteins in aqueous
solution by binding with one side to the hydrophobic part of the
protein and the other facing the surrounding water [37]. However,
many detergents do not maintain the structure and function of
membrane proteins, possibly due to the lack of lateral pressure or
due to the loss of “core lipids,” i.e., the tightly bound, quasiintegral lipids that are necessary for activity. Proteoliposomes are
model systems where membrane proteins are reconstructed into a
lipid bilayer after their extraction from the membrane with detergents, and this can already lead to an improved stability of membrane proteins in solution. More recently, membrane scaffold
proteins (MSPs) [38] and new types of polymers made of styrene–maleic acid (SMA) [39] or diisobutylene–maleic acid
(DIBMA) [40] have been used as they can keep the proteins in
their original lipid environment [41]. Since all these materials have
different characteristics, the choice of the optimal method for a
particular membrane protein cannot be predicted. Conditions for
efficient and functional solubilization need to be tested systematically using screening approaches (typically DSF or DLS) with different buffers and solubilizing molecules (Subheading 2.4).
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Bertrand Raynal et al.
assess the quality of the sample after storage, i.e., before it is used in
downstream applications. This can be done using a batch-to-batch
consistency protocol combining the basic QC tests described above
(Subheadings 1.1–1.3). It is essential that the protein’s physicochemical properties are not altered during storage. Processes such
as oxidation, precipitation, or (partial) degradation may impede the
study of protein structure and function or render it completely
impossible. This problem has been extensively studied for proteins
that are used as therapeutics, and it has been found that the most
crucial aspect in stabilizing the protein, and allowing storage for
18–24 months without loss of integrity and functionality, is the
final buffer composition; thus, it is worth investing time and consumables in the optimization of the buffer used for storage, which
may be distinct from that used downstream.
1.4.5 Membrane Protein
Buffer Optimization
Membrane proteins have large surface patches of hydrophobic
residues required for their insertion into or interaction with membranes. In aqueous solution, these patches promote protein aggregation through strong hydrophobic interactions. Thus, the
conditions for solubilizing membrane proteins are distinct from
those of cytoplasmic proteins. The most common method for
solubilizing (integral) membrane proteins is the addition of detergents or amphipols—polymers with a hydrophilic backbone decorated with hydrophobic sidechains, making them amphiphilic and
thus enabling them to stabilize membrane proteins in aqueous
solution by binding with one side to the hydrophobic part of the
protein and the other facing the surrounding water [37]. However,
many detergents do not maintain the structure and function of
membrane proteins, possibly due to the lack of lateral pressure or
due to the loss of “core lipids,” i.e., the tightly bound, quasiintegral lipids that are necessary for activity. Proteoliposomes are
model systems where membrane proteins are reconstructed into a
lipid bilayer after their extraction from the membrane with detergents, and this can already lead to an improved stability of membrane proteins in solution. More recently, membrane scaffold
proteins (MSPs) [38] and new types of polymers made of styrene–maleic acid (SMA) [39] or diisobutylene–maleic acid
(DIBMA) [40] have been used as they can keep the proteins in
their original lipid environment [41]. Since all these materials have
different characteristics, the choice of the optimal method for a
particular membrane protein cannot be predicted. Conditions for
efficient and functional solubilization need to be tested systematically using screening approaches (typically DSF or DLS) with different buffers and solubilizing molecules (Subheading 2.4).
20
Bertrand Raynal et al.