In many cases, high-level expression of recombinant proteins leads to the formation of protein aggregation commonly called as inclusion bodies. In order to obtain biologically active and soluble protein in high yield, inclusion bodies must then be solubilized and refolded in vitro.The first step is purification of inclusion bodies. For more information about protein refolding, you can refer to Overview of Protein Refolding.
Isolation and purification of inclusion bodies protein
Inclusion bodies can be pelleted by centrifugation for a relatively high density and cells are usually disrupted by high pressure homogenization (optionally following a lysosyme treatment). It is important that cell lysis is complete, because intact cells sediment together with the inclusion bodies, thus contaminating the preparation. After centrifugation, the pellet is washed with buffer containing either low concentrations of chaotropic agents (e.g. 0.5-1 M guanidine-HCl or urea) or detergents (e.g. 1% Triton X-100). This wash step is necessary to remove contaminants, especially proteins (proteases), that may have absorbed onto the hydrophobic inclusion bodies during processing.
Solubilizationof inclusion bodies protein
The washed inclusion bodies are resuspended and incubated in buffer containing a strong denaturant and a reducing agent (usually 20 mM DTT or β-mercaptoethanol). The addition of a reducing agent keeps all cysteines in the reduced state and cleaves disulfide bonds formed during the preparation. Optimal conditions for solubilization are protein specific and have to be determined for each protein.
Protocol of inclusion bodies purification
- Spin down cells from large culture at 6000 rpm for 20 min.
- Suspend the cell pellet (from 1L culture) in 30-35ml of PBST buffer. To facilitate lysis and inclusion body purification, add 0.5–1.0 % Triton X-100. Some EDTA and DTT, up to 50 mM, should be used in all subsequent steps to keep disulfides reduced.
- The cells can be lysed with either French press or sonication. With French press the cells should be passed at least twice through to ensure complete shearing of the genomic DNA. Sonication conditions should be optimised depending on the sonicator, tip, amount of cells in per volume of lysis buffer etc.
- Centrifuge cell lysate for approximately 20 min at 15,000 rpm, 4°C. Discard the supernatant.
- Wash the inclusion body pellets in a small volume of buffer containing 1-2% Triton X-100. This should solubilise membranes and membrane proteins. A short sonication (3 x 10 seconds) is very helpful during each wash step. Not only will it help to resuspend all the inclusion bodies, but it will also break unbroken cells and shear DNA.
- Centrifugation etc. as in 3.
- Solubilise the purified inclusion bodies into 6M guanidine HCl (Gnd-HCl) or 8M urea with appropriate buffer and 5-100mM DTT. Some inclusion bodies are very difficult to solubilise and you might want to leave them to dissolve over-night.
- Run a gel to check for the success of the inclusion body purification.
- Proceed to protein refolding.