Mempro™ Inclusion Body Purification Strategy for Membrane Protein

Creative Biolabs can provide advanced Mempro™ inclusion bodies purification services. We are capable of satisfying your specific demands all through your project.

The formation of bacterial inclusion bodies (IBs) is a main barrier for membrane protein production in the soluble and functional state, since excess membrane p proteins yielded from E. coli are subjected to form the misfolded, inactive and insoluble aggregates. It has been reported that most of recombinant membrane proteins expressed in E.coli are as inclusion bodies, which can also form in yeast, insect, and mammalian cells. Based on complex procedures, the active proteins can be reversible from inclusion bodies in vitro. This indicates that inclusion bodies can be dynamic constructions (noncrystalline and amorphous structures) formed by an unbalanced equilibrium between aggregated and soluble proteins in E. coli. It is therefore an exciting method to perform membrane protein production in inclusion bodies. While the challenge is not to purify the recombinant protein, but to solubilize and refold it into native and active protein.

Mempro™ Inclusion Bodies Purification Figure 1. Membrane protein purificaton from inclusion bodies.

Inclusion bodies containing insoluble proteins can be separated from cultured bacteria by homogenization, washing and centrifugation, which is then solubilized in denaturants, such as GuHCl or urea, for refolding the proteins. Notably, some reducing agents (such as DTT and GSSG) should be added to break up existing disulfide bonds, in order to obtain monomeric peptide chains (Figure 1).

Creative Biolabs can perform Mempro™ inclusion bodies protein purification using the most common protocol described as below：

Inclusion bodies preparation-Harvesting bacteria from culturation;
Inclsuion bodies dissoving-Selecting an appropriate buffer;
Protein refolding-Refolding by urea or rapid dilution.

Creative Biolabs can select detergent, urea (low concentration) or GuHCl to solublize the inclusion bodies based on your specific demands. It has been reported that these processes can dissolve about 60% of the insolube proteins. We have strong expertise in recovery of correctly refolded protein, and we can ensure the native conformation and structural integrity of the membrane proteins of interest.

Creative Biolabs also provides other various Mempro™ membrane protein production services. Please feel free to contact us for a detailed quote.

References

M. M. Carrio ́, et al. (2002). Construction and deconstruction of bacterial inclusion bodies. J. Biotechnol. 96(1): 3-12.
I. Palmer, et al. (2004). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr. Protoc. Protein Sci., Chapter 6: Unit 6 3.
R. Rudolph, et al. (1996). In vitro refolding of inclusion body proteins. Faseb. J., 10: 49-56.

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

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