In the cell there is competition between foldoing and aggregations.
In a many cases and in several host systems, recombinant proteins accumulate intracellularly in insoluble aggregates. The proteins in these so-called inclusion bodies are mostly inactive and denatured. In addition, dimers and multimers may be present. However, the expression of recombinant proteins in inclusion bodies can also be advantageous:
- the recombinant protein deposited in inclusion bodies can be 50% or more of the total cellular protein.
- the inclusion bodies often contain almost exclusively the overexpressed protein.
- in inclusion bodies the protein is protected from proteolytic degradation.
- expression in inclusion bodies will protect the cell against the toxicity of the recombinant protein.
The major problem is to recover biologically active and/or soluble protein in high yield. In order to accomplish this the protein in the inclusion bodies must by solubilized and refoldedin vitro. This procedure is carried out in three phases:
1. Isolation of inclusion bodies
Inclusion bodies have a relatively high density and, therefore, can be pelleted by centrifugation. 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 or 1 mg/ml sodium deoxycholate). This wash step is necessary to remove contaminants, especially proteins (proteases), that may have absorbed onto the hydrophobic inclusion bodies during processing.
2. Solubilization of aggregated proteins
The washed inclusion bodies are resuspended and incubated in buffer containing a strong denaturant and a reducing agent (usually 20 mM DTT or b-mercaptoethanol). The addition of a reducing agent keeps all cysteines in the reduced state and cleaves disulfide bonds formed during the preparation. Incubation temperatures above 30°C are typically used to facilitate the solubilization process. Optimal conditions for solubilization are protein specific and have to be determined for each protein. This is done most effectively by carrying out small-scale experiments (1-2 ml) to screen the different variables.
Important variables for solubilization are:
|Variable||good starting point|
|buffer composition (pH, ionic strength)||50 mM Tris-HCl, pH 7.5|
|incubation time||60 min|
|concentration of solubilzing agent||6 M guanidine-HCl or 8 M urea|
|total protein concentration||1-2 mg/ml|
|ratio of solubilizing agent to protein|
After solubilization the solution should be centrifuged to remove remaining aggregates which could act as nuclei to trigger aggregation during refolding. The best results are obtained when we use ultracentrifugation (30 min at >100,000 g).
3. Refolding of the solubilized proteins
Refolding of the solubilized proteins is initiated by the removal of the denaturant. The efficiency of refolding depends on the competition between correct folding and aggregation. To slow down the aggreagtion process refolding is usually carried out at low protein concentrations, in the range of 10-100 mg/ml. Furthermore, refolding conditions must be optimized for each individual protein. Important variables are:
- buffer composition (pH, ionic strength)
- additives (often in combination)
FoldIt, a commercial protein folding screen is available from Hampton Research.
If proteins contain disulfide bonds, the refolding buffer has to be supplemented with a redox system. The addition of a mixture of reduced and oxidized forms (1-3 mM reduced thiol and a 5:1 to 1:1 ratio of reduced to oxidixed thiol) of low molecular weight thiol reagent usually provides the appropriate redox potential to allow formation and reshuffling of disulfide bonds. The most commonly used redox shuffling reagents are reduced and oxidized glutathione, but also cysteine and cysteamine are used.
For certain protein, probably due to low solubility of folding intermediates, this procedure is not very effective. Alternatively, the protein is completely oxidized in the presence of a large excess of oxidized glutathione, followed by dilution in refolding buffer containing catalytic amounts of reduced glutathione.
Different methods for the refolding of proteins have been described:
Dialysis. The most used method is the removal of the solubilizing agent by dialysis. During dialysis the concentration of the solubilizing agent decreases slowly which allows the protein to refold optimally. The ratio of the volumes of the sample and the dialysis buffer should be as such that at the equilibrium concentration of the solubilizing agent the protein has completely refolded.
Slow dilution. The concentration of the solubilizing agent is decreased by dilution allowing the protein to refold. Usually the dilution is carried out slowly by step-wise addition of buffer or by continuous addition using a pump.
Rapid dilution. During dialysis and slow dilution the protein is exposed for an extended period of time to an intermediate concentration of the solubilizing agent (2-4 M urea or guanidine-HCl) where it is not yet folded but no longer denatured and thus extremely prone to aggregation. This could be prevented by the rapidly dilution of the solubilized protein solution into the refolding buffer. Aggregation during this process can be limited by adding mild solubilizing agents to the refolding buffer, such as non-detergent sulfobetaines.
Pulse renaturation. In order to keep the concentration of the unfolded protein low, thus limiting aggregation, aliquots of denatured protein are added at defined time points to the refolding buffer. The time intervals between two pulses have to be optimized for each individual protein.The process is stopped when the concentration of denaturant reaches a critical level with respect to refolding of the specific protein.
Chromatography. The solubilizing agent is removed using a chromatographic step. The application of different chromatography methods have been described:
- size exclusion chromatography (e.g. gel filtration on a Superdex 75 column)
- ion exchange chromatography
- affinity chromatography (e.g. IMAC using Chelating Sepharose or Ni-NTA agarose)
The denaturant is removed while the protein is slow migrating through the column or bound to the matrix. This usually gives a high yield of active protein even at protein concentrations in the mg/ml range.
Alternatively, it is possible to carry out chromatography under denaturing conditions before refolding the protein. Most modern chromatography resins are stable under the commonly used conditions for solubilization.
De Bernardez Clark, E. (1998) Refolding of recombinant proteins. Current Opinion Biotechnol. 9, 157-163.
Lilie, H., Schwarz, E. & Rudolph, R. (1998) Advances in refolding of proteins produced in E. coli. Current Opinion Biotechnol. 9, 497-501.