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0600 - Protein Refolding Kit
| Product number |
0600 |
| Product name |
Protein Refolding Kit |
| Quantity |
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| Supplier |
gentaur
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The information for protein folding is coded in the linear sequence of the polypeptide.2 With rare exception each protein can be denatured and refolded into a native active state under the right conditions. However, predicting the folding pathway for any given protein is a daunting challenge. For a 100 residue polypeptide there are 9100 accessible confi rmations. If each conformational search requires 10-15
seconds to complete it would take approximately 2.9 x 1079 years to examine each possible confi guration. This Levinthal paradox is resolved during protein folding by the progressive stabilization of intermediate states. Productive partially folded confi rmations are retained while non-productive folds are rearranged. The key appears to be the cooperative formation of stable native-like secondary structures
which serve to nucleate the process. In practical terms elucidating the folding pathway for any given protein requires painstaking analysis and signifi cant technical capabilities. Until a more thorough understanding of the relationship between primary protein sequence and structure is developed and the tools become available for in silico prediction of protein structure, the best available method for determining the conditions for protein folding remains empirical testing.
The parameter aff ecting protein refolding has been extensively reviewed.3,4,5 The key to successfully refolding a protein is to prevent off -pathway products from accumulating. These unwanted species form aggregates, a process which can be selfnucleating, resulting in poor recoveries of properly folded proteins. Intermediates with hydrophobic patches which are exposed to solvent are believed to play a
significant role in the formation of off -pathway products. Thus, to avoid off -pathway products the main tactic is a continuous or discontinuous buff er exchange where the renaturation buff er is designed to minimize these off pathway products.
The folding of proteins in solution is aff ected by a number of physiochemical parameters. These parameters include: Ionic strength, pH, temperature, oxidation state and protein concentration as well as the presence of hydrophobic, polar, chaotropic agents and other proteins. A comprehensive list is given by Clark4. Thus, the first step to develop a method for refolding proteins purifi ed from inclusion
bodies is to determine the composition of the refolding solution. The QuickFold™ Protein Refolding Screening Kit contains 15 diff erent buff er compositions which permit the rapid identifi cation of the factors which have a major eff ect on protein folding. From this information experiments can be performed to determine the optimum buffer formulation.
Five different techniques are employed to exchange the denaturant buff er with the refolding buff er including dilution, dialysis, diafi ltration, gel fi ltration and immobilization on a solid support. For screening purposes and, in some cases, small to moderate-scale production, dilution is the simplest approach. Its obvious drawback is that this technique leads to dilute protein solutions that would subsequently have to
be concentrated; with larger production volumes it would become cumbersome.
The other buff er exchange techniques are fully scalable to commercial production and can be performed under higher protein concentrations. Care must be taken to define the conditions which prevent aggregation under high protein concentrations. Several variations on the basic theme of buff er exchange have been noted for various proteins.
For example, a temperature leap in which the target protein is refolded at low temperature followed by a rapid increase in temperature to complete the process has been applied to the refolding of carbonic anhydrase II.6 During the low temperature incubation, folding intermediates which do not aggregate accumulate and upon a rapid temperature increase the fi nal product is formed with minimal misfolding.
Another approach is to expose the protein to intermediate denaturant concentrations that prevent the formation of aggregates but allow refolding to occur. This can be done by rapid dilution followed by slow dialysis into the fi nal buff er (example: lysozyme) or by gradually removing the denaturant by dilution during dialysis (example: immunoglobulin G).7 A general rule is that if a protein forms aggregates at
intermediate concentrations of denaturant, that a fast or slow dilution of denatured protein into renaturation buff er is best. If the protein does not form aggregates at intermediate denaturant concentrations, then slow dialysis with a gradual removal of the denaturant is best. |
| EUR | 484 | | USD | 604 | | GBP | 387 | | DKK | 3.604 | | JPY | 79.588 | | PLZ | 1.649 | | SEK | 4.520 | | NOK | 3.839 |
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