Pierce Protein Refolding Kit

Description

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.

Features of the Protein Refolding Kit:

• 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

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.

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".

Methods
Lysozyme was denatured overnight at 4°C in 8M 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 8M 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 0.4M 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, 2 mM GSH: 0.4 mM GSSG, and represents more than 90% of the solubilized lysozyme being refolded.

Specifications

Form:	Kit, Liquid, Powder
Product Line:	Pierce™
Product Size:	100 reactions
Reagent Type:	Chaotrope, Denaturant

Contents & storage

Upon receipt store at -20°C. Product may be stored at 4°C for up to two months. Product is shipped on dry ice.

Note:
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