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Misfolded proteins? Inclusion bodies?
Restore protein function with the Pierce Protein Refolding Kit
Recombinant DNA methodologies have paved the road for the production of large amounts of eukaryotic proteins in bacterial hosts. However, overproduction of proteins in transformed microorganisms often results in the formation of inactive, misfolded and insoluble protein aggregates called “inclusion bodies.” These misfolded proteins lack native functionality. Therefore, a reliable and cost-effective means for refolding proteins to their native state from inclusion bodies is of fundamental importance to basic research and biotechnological applications.
Paul Haney, Ph.D; Navid Haghdoost; and Connie Draveling;Pierce Milwaukee LLC
Refolding proteins can be a cumbersome and time-consuming task, as the refolding conditions have to be optimized for each respective protein. The Pierce Protein Refolding Kit is designed to facilitate this process and simplify the development of a high-yield/high-concentration refolding protocol.
Formulation and Design of the Pierce Protein Refolding Kit
The Pierce Protein Refolding Kit is based on the small molecule buffer additives methodology, a simple, common and historically successful method for initial refolding experiments.1-4 In addition, the Pierce Protein Refolding Kit is designed with a novel three-level adjustable matrix format that significantly reduces the amount of secondary optimization required, maximizes refolding yields and increases the ease of data interpretation. Buffer components are examined at three concentration levels, allowing a wide spectrum of folding conditions to be covered within one experiment, while an open design allows matrix conditions to be tailored to your target protein, preventing sample waste and unnecessary analysis.
Additionally, the Pierce Protein Refolding Kit comes with a
comprehensive Refolding Guide with details on 1) inclusion body isolation, solubilization and purification; 2) optimization of refolding conditions; and 3) analysis of refolding yields.
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Nine Base Refolding Buffers and seven additional buffer additives are supplied with the Pierce Protein Refolding Kit. The Base Refolding Buffers form a matrix covering a range of strong and weak denaturant conditions for the suppression of protein aggregation (Table 1). The supplied additives are used as secondary matrix factors, depending upon the protein type being refolded (Table 1 and Table 2). The set-up and handling of the Pierce Protein Refolding Kit is outlined in Figure 1.
Highlights:
- Adjustable matrix format – significantly reduces the amount of
secondary optimization required and increases the ease of data interpretation; known positive and negative interactions between buffer components are addressed, minimizing unnecessary analyses
- Robust – uses a fractional factorial matrix designed to evaluate a number of factors using proven folding reagent variables
• Convenience – conditions and components being examined are
limited to those having the most significant and general utility
as folding buffers
- High-purity reagents – reagents are formulated using stringent
standards
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Figure 1. Set-up and handling of the Pierce Protein Refolding Kit.
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| Table 1. Formulation and Matrix Design of the Pierce Protein Refolding Kit |
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Base Refolding Buffer*
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Factor 1
Guanidine**
(M)
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Factor 2
L-Arginine
(M)
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Factor 3
Additive 1
***
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Factor 4
Additive 2
***
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1
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0
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0
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1
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1
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2
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0
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0.44
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2
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2
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3
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0
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0.88
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3
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3
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4
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0.55
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0
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2
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3
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5
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0.55
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0.44
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3
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1
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6
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0.55
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0.88
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1
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2
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7
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1.1
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0
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3
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2
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8
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1.1
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0.44
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1
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3
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9
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1.1
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0.88
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2
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1
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*Each Base Refolding Buffer is supplied as a 1.1X stock solution. Each buffer stock contains the indicated denaturant concentrations as well as 55 mM Tris, 21 mM NaCl, 0.88 mM KCl adjusted to pH 8.2.
**Addition of solubilized protein sample will supply additional guanidine (GdnHCl).
***Factors 3 and 4 are defined by the researcher for customization of matrix conditions with the supplied additives (see Table 2). The numbers in the table refer to concentration levels that are specific for each additive. |
| Table 2. Buffer Additives |
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Buffer Additive
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Amount
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Polyethylene Glycol (PEG), 10 mM
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1 ml
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Dithiothreitol (DTT)
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100 mg
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Reduced Glutathione (GSH)
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120 mg
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Oxidized Glutathione (GSSG)
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100 mg
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EDTA, 100 mM
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1 ml
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MgCl2\CaCl2, 400 mM each
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0.5 ml
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CaCl2
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0.5 ml
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NaCI
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1 ml
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| Table 3. Results for refolding lysozyme using the Pierce Protein Refolding Kit |
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Trial No./ Base Refolding Buffer
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Guanidine*
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L-Arginine
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Redox Environment
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Protein Precipitation
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Relative Percent Recovery1,2
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1
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0.4 M
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0 M
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5 mM DTT
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YES***
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2%
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2
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0.4 M
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0.4 M
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2 mM GSH:0.2 mM GSSG
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YES**
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34%
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3
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0.4 M
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0.8 M
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2 mM GSH:0.4 mM GSSG
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NO
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81%
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4
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0.9 M
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0 M
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2 mM GSH:0.2 mM GSSG
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YES*
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47%
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5
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0.9 M
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0.4 M
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2 mM GSH:0.4 mM GSSG
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NO
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97%
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6
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0.9 M
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0.8 M
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5 mM DTT
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NO
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7%
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7
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1.4 M
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0 M
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2 mM GSH:0.4 mM GSSG
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NO
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100%
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8
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1.4 M
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0.4 M
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5 mM DTT
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NO
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7%
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9
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1.4 M
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0.8 M
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2 mM GSH:0.2 mM GSSG
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NO
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7%
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Key:
* Indicates the amount of protein precipitation that occurred (** = low, *** = high).
- A minor amount of refolding may occur during activity assay.
- Recovery is reported as a percentage of the trial (# 7) with highest activity after refolding. Compared to non-denatured controls, Trial 7 represents greater than 90% of the solubilized lysozyme refolding.
- Each buffer contains the indicated denaturant and redox concentrations as well as 50 mM Tris, 18 mM NaCl, 8 mM KCl, 1 mM EDTA; pH 8.2.
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Methods
Lysozyme was denatured overnight at 4°C in 8 M GdnHCl, 10 mM DTT, 50 mM Tris, pH 8.0 at 20 mg/ml. Reduced glutathione, oxidized glutathione and DTT were added to refolding buffers as determined by the matrix layout (Table 3). All solutions were equilibrated to 4°C. Immediately before adding solubilized lysozyme to refolding buffers, the DTT was removed using a protein desalting spin column equilibrated in 8 M GdnHCl, 50 mM Tris; pH 8.0. Lysozyme was then added to the refolding buffers at a final concentration of 1 mg/ml. This addition supplies 0.4 M GdnHCl to the Base Refolding Buffers. Refolding was allowed to proceed for 18 hours at 4°C. Refolding yields were determined by measuring lysozyme activity with the EnzChek Lysozyme Assay Kit (Molecular Probes) using a Tecan SPECTRAFluor Plus System.
Results and Discussion
The basic protocol for protein refolding requires that inclusion bodies are first isolated, purified and then solubilized with a strong denaturant, such as guanidine hydrochloride (GdnHCl), to produce a completely unfolded protein. The solubilized protein is then diluted or dialyzed into a refolding buffer to reduce the denaturant concentration, allowing the protein to refold based on the information contained in its primary sequence. When using optimized conditions many proteins can be reliably refolded at concentrations >1 mg/ml. However, if the denaturant is removed and replaced with a non-optimized refolding buffer, protein aggregation strongly competes with renaturation and only minimal amounts of native protein are recovered. The degree of aggregation that occurs during refolding is largely dependent on protein concentration, concentration of strong and weak denaturants, pH, temperature, and the redox environment. Ionic strength, divalent cations, polymers and cofactors can also promote refolding of some proteins.
The results for refolding reduced and denatured lysozyme using the Pierce Protein Refolding Kit are reported in Table 3. For this example we treated lysozyme as if the presence of disulfide bonds in the native state was unknown. Reformation of native lysozyme was suppressed at the lowest and highest denaturant concentrations present within the protein refolding matrix (trial numbers 1, 2 and 9). Refolding was also suppressed by the presence of DTT (trial numbers 1, 6 and 8), showing the importance of reforming disulfide bonds in the folding of lysozyme. Highest lysozyme activity regained in the experiment was achieved in trial seven, which contained 1.4 M GdnHCl, 0 M L-arginine, 2 mM GSH: 0.4 mM GSSG, and represents more than 90% of the solubilized lysozyme being refolded.
References
- Rudolph R. and Lilie, H. (1996). In vitro folding of inclusion body proteins. FASEB 10:49-56.
- Lilie, H., et al. (1998). Advances in refolding of proteins produced in E. coli. Curr. Opin. in Biotech. 9:497-501.
- Middelberg, A. (2002). Preparative protein refolding. Trends Biotechnol. 20(10):437-443.
- Hevehan, D.L. and Clark, E.D.B. (1997). Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54(3):221-230.
Ordering Information
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 Certificate of Analysis  Product Instructions  MSDS |
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Product # |
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Pkg. Size |
Files |
Price |
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89867 |
Pierce Protein Refolding Kit
| Sufficient components to conduct 100 1 ml refolding experiments | | Kit contains: | | Base Refolding Buffer 1 | 10 ml | | Base Refolding Buffer 2 | 10 ml | | Base Refolding Buffer 3 | 10 ml | | Base Refolding Buffer 4 | 10 ml | | Base Refolding Buffer 5 | 10 ml | | Base Refolding Buffer 6 | 10 ml | | Base Refolding Buffer 7 | 10 ml | | Base Refolding Buffer 8 | 10 ml | | Base Refolding Buffer 9 | 10 ml | | Dithiothreitol(DTT) | approximately 100 mg* | | Reduced Glutathione (GSH) | approximately 120 mg* | | Oxidized Glutathione(GSSG) | approximately 100 mg* | | 10 mM Polyethylene Glycol(PEG) | 1 ml | | Divalent Cation Stock | 0.5 ml | | 5 M NaCl | 1 ml | | 100 mM EDTA | 1 ml | | *Note: Dry components are dispensed by volume – and not by weight. |
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Kit |
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$320.00 |
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