Over expression of recombinant protein in bacteria can lead to the accumulation of insoluble protein, which aggregates and is sequestered to inclusion bodies. Slow growth rate and low temperatures during protein expression will help in solubility of many over expressed proteins, however some proteins still form Inclusion Bodies. Inclusion Bodies are pure proteins aggregated in bacterial cytoplasm, but sometimes can be formed in periplasm. Though the process of purification is tedious, we can get pure active protein by using various purification steps. Utmost care to be taken during Inclusion Bodies purification as the process is harsh on delicate proteins and may result in the loss of protein activity.
The first and crucial step in Inclusion Bodies solubilization is obtaining pure Inclusion Bodies from cell pellet. During the lysis procedure, both chemical and physical methods can be used. Lysosyme, non- ionic detergents, like Triton X-100 and Tween-20, can be added to the lysis buffer and physical methods like sonication, high pressure disruption can be used. There is a need of including additional steps to wash the Inclusion Bodies thoroughly so that one can get pure Inclusion Bodies pellet before solubilization. Impure Inclusion Bodies result in irreversible protein aggregation during refolding step and loss of protein activity.
Wash the Inclusion Bodies with high amounts of non-ionic detergents (For example 2% Triton X-100) or low amounts of denaturants (2M Urea) to remove contaminant proteins. Note that if the weight of Inclusion Bodies pellet is 1/10th of the weight of wet cell pellet it indicates pure protein in Inclusion Bodies. Continue to Inclusion Bodies solubilization by trying combination of different buffers and denaturants. For ease of use one can try this kit from G-Biosciences, IBS buffer kit.
Once the Inclusion Bodies is solubilized the next important step is to refold the protein. Solubilised protein attains linear inactive form, this protein, when refolded properly, attains folded active form. The refolding step is very important for active pure proteins are desired for various applications. During refolding the denaturants are slowly removed by one of the methods listed.
- Pulse refolding method is one of the widely used method where small amounts of concentrated denatured protein was added to a large volume of refolding
- Dialysis method where denatured protein is dialysed against large volumes of refolding buffer until the denaturant is
- Dilution method where refolding buffer was slowly added to the concentrated denatured
- Column chromatography under denaturing conditions, where the denatured protein is passed through either IMAC column or desalting
During refolding, protein should be maintained at lower concentrations (0.01mg - 0.1 mg/ml). Once the process is done it is very important to find out whether the protein is correctly refolded or not. By using enzyme activity assay we can check whether the protein refolding is successful. Though not all proteins can be checked by this, some techniques like size exclusion chromatography, Native PAGE, Circular Dichroism (CD) can also be used.
References:
- Protein Expression and Refolding – a practical guide to getting the most out of inclusion bodies”, Cabrita and Bottomley, 2004, Biotechnology Annual Review, 10:31-50.
- Palmer I, Wingfield Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli. Current protocols in protein science / editorial board, John E Coligan . [et al]. 2004;CHAPTER:Unit-6.3. doi:10.1002/0471140864.ps0603s38.
