Membrane Protein Purification

Purification is almost an unskippable step during protein preparation. Many scientists already have some fundamental skills for purifying water-soluble proteins. However, unlike water-soluble proteins, membrane proteins have to be handled with extra cautions and purifying them can be rather technically challenging. The reason is that most membrane proteins are embedded or associated with cell membranes, and it may be difficult to find a suitable medium that plays the same role as cell membranes to stabilize them in their native form. Therefore, in contrast to soluble proteins, unwanted folding, conformational changes, and aggregation frequently occur during membrane protein expression, which will lead to not only a huge reduction in yield but also damages in protein structure and functionality.

Membrane Protein Purification (Creative Biolabs)

Affinity Tag in Membrane Protein Purification

The position of a specific tag may affect expression level, behavior in solution and even protein function. Furthermore, if the protein N-terminus may be subject to signal peptidase cleavage, then the affinity tag needs to be located elsewhere. A general rule of thumb for membrane protein tags is to place them at solution accessible region, either N- or C-terminal or even cytoplasmic loop of the target protein. Most commonly used tags include Poly-histidine tag, Strep tag II, FLAG tag, Rho1D4-tag, GST-tag, etc.

Affinity Tag in Membrane Protein Purification (Creative Biolabs)

Sometimes it is required to remove the affinity tag for further study of the target protein. Hence, protease sites are constructed right upstream of the tag sequence. FLAG tag conveniently encodes the enterokinase protease site DDDDK and this protease works well in detergent-containing buffer. Thrombin is also recommended for removal of tags from membrane proteins as this protease shows reliable activity in many detergents. In contrast, tobacco etch virus (TEV) protease may suffer from poor activity in several common detergents such as octyl-glucopyranoside.

No matter whether your membrane proteins are tagged or non-tagged, helical or barrel, single-span or multi-span, single proteins or large protein complexes, and in a small quantity or an industrial-scale quantity, this comprehensive screening platform can always help you find the optimal protocol and condition. Our broad expertise covers purifications of GPCRs, ion channels, transporters, membrane enzymes, membrane fusion proteins, etc. Depending on different features of the target membrane proteins, the following chromatographic methods can be chosen at Creative Biolabs individually or in combination if needed:

Affinity [depending on the tag(s) added to your membrane proteins]
Ion-exchange (cation or anion)
Gel-filtration/size-exclusion
Hydrophobic interaction

Creative Biolabs will check the protein homogeneity using SDS-PAGE, western blot, or other methods to confirm that the desired purity is achieved. Creative Biolabs will always work on a schedule adapted to the progress of your projects. Please do not hesitate to inquire us for more information.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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