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Pierce Protein Refolding Kit


Discover conditions for refolding solubilized, denatured recombinant proteins.

Pierce Protein Refolding Kit

The Thermo Scientific Pierce Protein Refolding Kit includes high-purity reagents and detailed instructions for using a matrix strategy to determine optimal buffer conditions for refolding recombinant proteins that have been denatured and solubilized from inclusion bodies.

The kit contains the essential reagents and complete strategy for determining optimal buffer conditions to refold denatured recombinant proteins to restore native structure and function. Nine base refolding buffers form a matrix that includes a range of strong and weak denaturant conditions for the suppression of protein aggregation. The supplied additives are used as additional matrix factors, depending on the protein type being refolded. Buffer components are examined at three concentration levels, allowing a wide spectrum of folding conditions to be tested within one experiment. The adjustable design allows matrix conditions to be tailored to the target protein, preventing sample waste and unnecessary analysis, while maximizing refolding yields. The Pierce Protein Refolding Kit is accompanied by a comprehensive Refolding Guide with details on isolating, solubilizing and purifying inclusion bodies; optimizing refolding conditions; and analyzing refolding yields.

Highlights:

  • Robust – conditions and components examined are limited to those having the most significant and general utility as folding buffers
  • Convenient – three-level matrix design significantly reduces the amount of secondary optimization required and increases the ease of data interpretation
  • Adjustable matrix format – allows refolding experiments to be customized to the target protein; known positive and negative interactions between buffer components are addressed, minimizing unnecessary analyses
  • High-purity reagents – reagents are formulated using stringent standards so that consistent results are attained

Product Details:

Note: The following section is based on the original Previews article that reported on the development of the Pierce Protein Refolding Kit, which was originally called "Pro-Matrix".

Misfolded proteins? Inclusion bodies? Restore protein function with the Pierce Protein Refolding Kit

Paul Haney, Ph.D; Navid Haghdoost; and Connie Draveling

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.

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 (Refs. 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.

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.

Protocol and strategy for protein refolding optimization.
Figure 1. Set-up and handling of the Pierce Protein Refolding Kit.

 

Table 1. Formulation and Matrix Design of the Pierce Protein Refolding Kit. Each Base Refolding Buffer is supplied as a 1.1X stock solution. Each buffer stock contains the indicated denaturant concentrations as well as 55mM Tris, 21mM NaCl, 0.88mM 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.
Base Refolding Buffer Factor 1
Guanidine (M)
Factor 2
L-Arginine (M)
Factor 3
Additive 1
Factor 4
Additive 2
1 0 0 1 1
2 0 0.44 2 2
3 0 0.88 3 3
4 0.55 0 2 3
5 0.55 0.44 3 1
6 0.55 0.88 1 2
7 1.1 0 3 2
8 1.1 0.44 1 3
9 1.1 0.88 2 1

 

Table 2. Buffer Additives
Buffer Additive Amount
Polyethylene Glycol (PEG), 10 mM 1mL
Dithiothreitol (DTT) 100mg
Reduced Glutathione (GSH) 120mg
Oxidized Glutathione (GSSG) 100mg
EDTA, 100 mM 1 mL
MgCl2 and CaCl2, 400mM each 0.5mL
CaCl2 0.5mL
NaCI 1ml

 

Table 3. Results for refolding lysozyme using the Pierce Protein Refolding Kit. Each buffer contains the indicated denaturant and redox concentrations as well as 50mM Tris, 18mM NaCl, 8mM KCl, 1mM EDTA; pH 8.2. Recovery is reported as a percentage of the trial (Buffer 7) having highest activity after refolding. Compared to non-denatured controls, Trial 7 represents greater than 90% of the solubilized lysozyme refolding. A minor amount of refolding may occur during activity assay.
Buffer Guanidine L-Arginine Redox Environment Protein Precipitation Relative
Percent Recovery
1 0.4M 0M 5mM DTT
 
YES (high) 2%
2 0.4M 0.4M 2mM GSH
0.2mM GSSG
YES (mid) 34%
3 0.4M 0.8M 2mM GSH
0.4mM GSSG
NO 81%
4 0.9M 0M 2mM GSH
0.2mM GSSG
YES (low) 47%
5 0.9M 0.4M 2mM GSH
0.4mM GSSG
NO 97%
6 0.9M 0.8M 5mM DTT
 
NO 7%
7 1.4M 0M 2mM GSH
0.4mM GSSG
NO 100%
8 1.4M 0.4M 5mM DTT
 
NO 7%
9 1.4M 0.8M 2mM GSH
0.2mM GSSG
NO 7%

Methods

Lysozyme was denatured overnight at 4°C in 8M GdnHCl, 10mM DTT, 50mM Tris, pH 8.0 at 20mg/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 8M GdnHCl, 50mM Tris; pH 8.0. Lysozyme was then added to the refolding buffers at a final concentration of 1mg/mL. This addition supplies 0.4M 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(tm) Lysozyme Assay Kit (Molecular Probes) using a Tecan(tm) 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 1.4M GdnHCl, 0M L-arginine, 2mM GSH: 0.4mM GSSG, and represents more than 90% of the solubilized lysozyme being refolded.

References

  1. Rudolph R. and Lilie, H. (1996). In vitro folding of inclusion body proteins. FASEB 10:49-56.
  2. Lilie, H., et al. (1998). Advances in refolding of proteins produced in E. coli. Curr. Opin. in Biotech. 9:497-501.
  3. Middelberg, A. (2002). Preparative protein refolding. Trends Biotechnol. 20(10):437-443.
  4. Hevehan, D.L. and Clark, E.D.B. (1997). Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54(3):221-230.

Related Products:

Inclusion Body Solubilization Reagent
8M Guanidine-HCl Solution
B-PER Bacterial Protein Extraction Reagents
 


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
89867 Pierce Protein Refolding Kit
Formulation: Multi-component kit
Sufficient For: 10 full-matrix optimization experiments

Kit Contents:
Base Refolding Buffers 1 to 9, 10mL ea.
Dithiothreitol (DTT), 100mg
Reduced Glutathione (GSH), 120mg
Oxidized Glutathione (GSSG), 100mg
Polyethylene Glycol (PEG), 10mM, 1mL
Divalent Cation Solution, 0.5mL
NaCl, 5M, 1mL
EDTA, 100mM, 1mL
Refolding Guidebook
100-rxn kit Product Instructions for product #89867 Pierce Protein Refolding Kit MSDS for product #89867 Pierce Protein Refolding Kit Certificate of Analysis for product #89867 Pierce Protein Refolding Kit $410.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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