Conditioning Purified Membrane Proteins

Extracted from Purifying Challenging Proteins - Principles and Methods, GE Healthcare, 2007

The purified membrane protein often has to be concentrated or transferred to a suitable environment for further analysis. Common manipulations include concentration, desalting, buffer exchange, detergent exchange, and detergent removal. Protocols for these operations are given below.

Concentration

Ultrafiltration-based concentration techniques are commonly used for membrane proteins. They often also concentrate the detergent micelles and hence change the detergent:protein ratio, which may lead to precipitation. Another potential problem is an increased protein concentration at the filter surface which may lead to aggregation and precipitation. Column chromatography is an alternative, milder concentration technique. IMAC is excellent for concentration of histidine-tagged proteins, and ion exchange chromatography is used for both tagged and non-tagged proteins. Small diameter (10 to 35 µM) and monodisperse chromatography beads are advantageous to use because they minimize band broadening at elution. Desalting may be needed to remove imidazole or salt after column concentration.

HisTrap columns can be used for concentration of purified histidine-tagged membrane proteins under mild conditions. The purified protein is adsorbed at neutral pH in a buffer containing 0 to 20 mM imidazole. Several mg of protein can usually be adsorbed, but the maximum column capacity is protein dependent. Elution is achieved in a single step by elution with 0.5 to 1 M imidazole, as required.

Ion exchange columns (e.g., HiTrap Q HP, RESOURCE Q, or Mono Q 5/50 GL) can be used for concentration of membrane proteins under mild conditions. Elution is achieved in a single step with high salt (e.g., 0.5 to 1 M NaCl) or a change in pH. Protocol details will depend on the specific protein.

Desalting or buffer exchange

Desalting protocols for membrane proteins use detergents in buffers but are otherwise identical to those for soluble proteins.

Material

Sample volume per run (mL)	Product	Operation
0.01 to 0.1	illustra™ MicroSpin™ G-25 Columns	Centrifuge (spin column)
1.5 to 2.5	Disposable PD-10 Desalting Columns	Manual (gravity feed)
0.25 to 1.5	HiTrap Desalting	Chromatography system, a peristaltic pump or a syringe (sample volume can be increased to 3 or 4.5 mL when using two or three columns, respectively, connected in series)
2.5 to 15	HiPrep 26/10 Desalting	Chromatography system (sample volume can be increased to 30, 45, or 60 mL when using two, three, or four columns, respectively, connected in series)

Procedure

Follow the recommended procedure supplied with each product. The general outline is described below.

1. Equilibrate the column with the desired buffer.
2. Apply the sample. The maximum recommended sample volume should not be exceeded.
3. Apply elution buffer and collect the protein material in recommended fraction volumes.

Detergent exchange

Detergent exchange may be needed for a number of reasons. Both for cost reasons and performance aspects, it is common that the detergent used for solubilization and/or purification is exchanged for a detergent that is more suitable for further analyses.

Dialysis is often used for this purpose but can be very inefficient. Detergents with a low CMC are dialyzed very poorly since micelles pass slowly or not at all through the dialysis membrane. Ultrafiltration-based approaches can be inefficient for the same reason. In addition to problems with permeability, the protein concentration may become very high close to the dialysis/ultrafiltration membrane, which may lead to extensive aggregation.

Column-based approaches for detergent exchange are often mild and efficient. A potential drawback with column-based methods is that the elution buffer may contain unwanted components that need to be removed from the eluted membrane protein. This problem can be solved by desalting and buffer exchange as described in the previous section.

Detergent exchange of purified membrane proteins with Q Sepharose Fast Flow or RESOURCE Q

Material

Column choices:

HiTrap Q FF, 1 mL

HiTrap Q FF, 5 mL

RESOURCE Q, 1 mL

RESOURCE Q, 6 mL

Procedure

Use the flow rate recommended in the column instructions.

The buffer conditions of the sample and binding buffer (ionic strength and pH) that allow binding need to be determined for each protein.

1. Equilibrate the column with the 2 CV of a low ionic strength buffer, containing the same detergent and detergent concentration as the protein sample. A suitable buffer is 50 mM TrisHCl, 50 mM NaCl, pH 8.0, plus detergent.
2. Apply the sample.
3. Wash the column with 5 to 12 CV of a low ionic strength buffer containing the new detergent. A suitable buffer is 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, plus the new detergent.
4. Elute in one step with the same buffer containing 0.6 M NaCl. A higher salt concentration can be used if the protein is not quantitatively eluted.

Avoid using anionic detergents with this protocol.

HisTrap HP, 1 mL can be used for detergent exchange of purified histidine-tagged membrane proteins following a similar protocol as for Q Sepharose Fast Flow above. Elution is achieved in a single step by elution with 0.5 to 1 M imidazole.

Detergent exchange with Phenyl Sepharose

Detergent saturated Phenyl Sepharose is used to exchange detergents for one another in the presence of membrane proteins. Protein binding to the medium is minimized by using a high pH.

Material

Column choices:

HiTrap Phenyl FF (high sub), 1 mL

HiTrap Phenyl FF (low sub), 1 mL

HiTrap Phenyl HP, 1 mL

Buffer: 20 mM Tris-HCl, 1 mM EDTA, appropriate detergent at or slightly above the CMC, pH 8 to 9

Sample: 1 to 4 mg membrane protein/mL, in 0.5 to 1 mL of buffer and the detergent to be removed.

Procedure

1. Saturate the column with the new detergent in buffer. Detergent concentration should be at or slightly above the CMC.
2. Apply the sample to the column and elute with buffer (isocratic elution). The detergent in the sample displaces detergent that had been adsorbed to the column.

For membrane proteins that tolerate organic solvents, detergent can be removed using polymer-based reversed phase chromatography (RPC) media (e.g., RESOURCE RPC, 1 mL). The purified, detergent-solubilized membrane protein is adsorbed to the column, washed with a weak phosphate buffer (without detergent) and eluted with acetonitrile. The eluted protein can be lyophilized and reconstituted.