Renaturation Basic Kit for Proteins

Introduction

Overexpression of proteins in prokaryotes often leads to the production of insoluble aggregates of misfolded proteins in inclusion bodies. These misfolded proteins lack their desired natural functionality. Often considered a nuisance, the formation of inclusion bodies has also some advantages:

• high enrichment of desired protein
• proteins are protected against protease acivities

Regaining the desired functionality requires correct refolding of proteins, which can only be achieved under certain specific conditions. The Renaturation Basic Kit for Proteins (96827) is a rapid empirical screening method used to determine the best conditions for the renaturation of proteins which have been solubilized from inclusion bodies. Its benefits are:

• Convenient screening method based on ready made solutions
• Reliable high quality of reagents used for preparation (BioChemika Ultra quality grade)
• Conditions cover defined pH range of 6.5 to 8.5, defined redox potential and ionic strength conditions, as well as the effect of a carefully selected detergent
• Larger volumes of components are available

The Kit contains 50 different ready-to-use solutions. All solutions are sterile filtered using 0.22 micron filters and are available separately as 100 ml bottles. The storage of the Kit should take place at 2 - 8 °C

Technical Bulletin: Application

The Renaturation Basic Kit for Proteins is a rapid empirical screening method used to determine the best conditions for the renaturation of proteins which have been solubilized from inclusion bodies. It is known that physical parameters, such as pH, redox potential, ionic strength, low-molecular weight additives and temperature, may have an effect on the efficiency of in vitro renaturation processes.^1-4 Screening with this kit is an easy method to find renaturation conditions in a defined pH range of 6.5 to 8.5, defined redox potential and ionic strength conditions, as well as the effect of one sulfobetaine, namely 3-(1-1-pyridinio)-1-propanesulfonate. Other parameters, e.g., concentration of the solubilized protein, temperature and additional additives, can easily be added to the basic screening conditions of this kit.

Overexpression of proteins in prokaryotes often leads to the production of insoluble aggregates of misfolded proteins in inclusion bodies. Often considered a nuisance, the formation of inclusion bodies has the advantage of a high enrichment of the desired protein at an early stage of purification. Furthermore, the recombinant protein is protected in inclusion bodies against proteolysis by intracellular proteases. These inclusion bodies can easily be purified and may be the best method for the production of proteins that are lethal to the host cells. However, the solubilization of the expressed protein can only be obtained using strongly denaturing conditions. The major task is to achieve an efficient in vitro renaturation to the properly folded protein.

Formulation/Storage/Stability

All reagents are formulated using high purity reagents, mostly FLUKA BioChemika Ultra, and ultrapure water. BioChemika Ultra chemicals have been used successfully for different crystallization methods and other applications that are highly sensitive to impurities. All solutions are sterile filtered using 0.22 micron filters and are available separately as 100 ml bottles. Larger quantities are available on request. The reagents are stable at room temperature if the bottles aren’t opened. To enhance reagent stability, it is strongly recommended that kit reagents be stored at 2-8 °C or -20 °C. Do not expose the reagents to ultraviolet light.

If the samples contain phosphate, borate, or carbonate buffers, inorganic crystals may form when using those reagents containing divalent cations, e.g., magnesium, calcium, or zinc. To avoid false positives, use phosphate, borate, or carbonate buffers at concentrations of 10 mM or less, or replace the phosphate, borate, or carbonate buffers with a more soluble buffer that does not complex with divalent cations.

Sample Preparation

Since inclusion body proteins do not readily disintegrate under physiological conditions, the solubilization requires rather strong denaturants such as 6 M guanidine hydrochloride (BioChemika Ultra Quality) or 6 – 8 M urea (51457, BioChemika Ultra). Guanidine hydrochloride is usually preferred over urea because it is a rather strong chaotropic agent that may solubilize extremely sturdy inclusion bodies, and because urea solutions may contain isocyanate leading to carbamylation of the free amino groups of the polypeptide. If urea is used for inclusion body solubilization, scavengers containing free amino groups should be included in the solubilization cocktail. In the case of proteins containing cysteine, the isolated inclusion bodies usually contain some interchain disulfide bonds which reduce the solubility. Addition of reducing agents, like low-molecular weight thiol reagents, in combination with chaotropic agents allows reduction of the interchain disulfide bonds.

The degree of purification of the proteins depends on the intended use. Because inclusion bodies collected after cell disruption are usually relatively homogeneous, the proteins can be renatured directly after solubilization without further purification. Rigorous purification of the inclusion body protein may not be required for efficient refolding. If the influence of other proteins like chaperones or other folding enhancers is of interest, or if spectroscopic techniques are used to monitor in vitro folding, purification of the solubilized inclusion body protein may be necessary.

The solubilization of the Inclusion bodies is a prerequisite for the application of this renaturation kit and is usually achieved by using 6M guanidine hydrochloride solution. The solubilization can itself be optimized, but as a starting point the following procedure can be useful.

1. The pellet from 1 l bacterial suspension is resuspended in 20 ml 50 mM HEPES-NaOH, pH 7.5, 0.5 M NaCl (71378), 1 mM PMSF (78830), 5 mM DTT (43817) and 0.35 mg/ml lysozyme (62970), then incubated for 30 min at 20 °C.
2. 200 ml Triton X-100 (93418) is added and then sonicated until the solution clears.
3. The extract is treated with 20 mg/ml DNAse I (31132) at 37 °C and the inclusion bodies are centrifuged at 30’000 g for 30 min at 4 °C.
4. The pellet is washed twice with TBS (93312) or PBS (79378) with 1% Triton X-100 and centrifuged at 30’000 g for 30 min at 4 °C.
5. The pellet ( inclusion bodies) is solubilized in 2 ml 50 mM HEPES-NaOH, pH 7.5, 6 M guanidine hydrochloride, 25 mM DTT and incubated for 1hr at 4 °C.
6. Insoluble material is removed by centrifugation at 100’000 g for 10 min. It is important to remove existing aggregates that can act as nuclei to start aggregation during renaturation.
7. Determine the protein concentration and adjust to 1 mg/ml using 50 mM HEPES-NaOH, pH 7.5, 6M guanidine hydrochloride, 25 mM DTT and proceed directly to renaturation.

Procedure

The following describes one way of using the Renaturation Basic Kit for Proteins. Note: It is important to mix the samples quickly and thoroughly.

Prepare plates with 24 wells.

1. Using a clean pipet tip for each reagent, pipet 900 ml of cold reagent 1 into the first well, A1.
2. Add 100 ml of the solubilized protein sample into the well. Mix the solubilized protein as quickly as possible by aspirating and dispensing. The final protein concentration should not exceed 0.1 mg/ml. Keep the tip in the drop during mixing to avoid foaming.
3. Repeat 1. and 2. using the remaining reagents.
4. Seal the entire plate with clear sealing tape.
5. The screening can be performed in duplicate at different temperatures if sample quantities permit. lncubate and store the plates in a place with stable temperature.
6. Determine yields of renaturation after 1 hr and 24 hr of renaturation by using a reliable, fast and easy assay to monitor native structure formation: enzymatic activity, HPLC, spectroscopy, ligand binding, ELISA or bioassays. Measuring the solution turbidity is a good indication of protein aggregation.
7. Compare the observations between the sample in the different reagents, and at the different incubation temperatures.

The suggested reagents (substances, concentration) are starting points and have to be optimized in order to obtain highest renaturation yields.

Interpreting Results

In order to interpret the renaturation yield, a quantitative measure of the function of the properly folded pure protein, e.g. maximum specific enzyme activity is needed.

This kit allows a quick screen to determine whether the protein of interest can be renatured. After successful renaturation from inclusion bodies, the kit gives some insight into which of the factors are most important and this information can be used to refine the renaturation conditions. Key parameters are protein concentration, residual guanidine concentration and temperature. If the results of the screen indicates that the renaturation from inclusion bodies is not feasible, additional auxiliary reagents should be screened which favor the formation of the native fold and minimize the aggregation of folding intermediates. A broad range of such reagents, e.g., arginine, PEG, sulfobetaines, detergents and chaotropes, are available from our FLUKA (see our FLUKA / Riedel-de Haën Catalog). It is recommended that reagents with the highest purity are used to minimize aggregation and to increase the yield of the properly folded protein.

References

1. R. Rudolph & H. Lilie, The FASEB Journal, 10, 49 (1996)
2. L. Vuillard, T. Rabilloud & M.E. Goldberg, Eur. J. Biochem. 256, 128 (1998)
3. P.H. Bessette, F. Aslund, J. Beckwith & G. Georgiou, PNAS 96, 13703 (1999)
4. N. Armstrong, A. DeLencastre & E. Gouaux, Protein Science 8,1475 (1999)

Click here for taking a look at the observation sheet

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