Protein Refolding Kit, BioAssay(TM)is a screening kit that enables researchers to pinpoint the critical factors for refolding their protein in as little as 1 hour. Unlike many traditional methods, this kit employs a fractional factorial matrix design that allows the researcher to screen their specific protein in 15 different buffers quickly and easily. Researchers are able to examine a wider range of conditions all within a single experiment, simplifying the process of identifying the best buffer composition and method for the refolding of a given protein. The kit comes with enough buffer for 10 refolding experiments, as well as supplemental dithiothreitol and a Glutathione Redox System. Each buffer is available for individual purchase and is supplemented with the necessary Glutathione Redox System and/or DTT. Individual buffers come in 500ml and 1000ml amounts.
Protein Refolding Kit, BioAssay(TM) is designed to help simplify the process of identifying the buffer composition and method best suited for protein refolding.
For more than 20 years E. coli has proved to be a reliable host for the production of heterologous proteins. The well defined genetics, readily available host-vector systems, and established methods has made E. coli the first choice for the expression of recombinant proteins. Despite the history of successes, the expression of heterologous proteins the production of soluble functional protein remains unpredictable. Frequently, the over expression of a protein in E. coli results in the formation of insoluble inclusion bodies.
The reasons for inclusion body formation are not fully known. Since translation is a slower process than protein folding, it is likely that the misfolding of translation intermediates plays some role. Posttranslational modification, such as glycosylation and lyposylation, are known to affect the secondary structure of proteins. In bacteria, these modifications are mostly absent. Further, the chemical environment in which translation occurs in the eukaryotic cell is different than that of the bacterial cell. Each of these factors contributes to varying degrees to how the nascent polypeptide folds, or in the case of recombinant protein expression, misfolds (1).
Several approaches have been used to mitigate misfolding during the over expression of proteins in E. coli. These include:
1. Fusion of the target protein with a more soluble partner, typically a bacterial protein;,
2. Co-expression of folding catalysts and chaperones;
3. Expression under cultures conditions which reduce the translation rates or effect the intracellular environment;
4. Modification of the protein sequence. Each approach has advantages and disadvantages which must be weighed in light of the intended end-use of the target protein. Further, not all proteins respond favorably to any given approach. Again which approach is best suited to a given protein must be determined empirically and success in producing and recovering soluble active protein is not guaranteed.
Both a bane and blessing, the formation of inclusion bodies renders the expressed protein unusable. The purification of a protein as an inclusion body is relatively simple, easily scalable for commercial applications, and in many cases, can stabilize the protein until a sufficient degree of purity is obtained. The challenge is that the protein must then be recovered from the insoluble particle. The recovery of soluble active protein from purified inclusion bodies requires the denaturation of the polypeptide and then its refolding to an active form. Many examples of proteins recovered from inclusion bodies are well known and used for both commercial and academic applications. There are well established methods for purifying inclusion bodies and solublizing the aggregated protein by denaturation. There is, however, no reliable method for predicting the conditions needed to refold the protein. Thus, the identification of the conditions needed to properly refold the protein remains an empirical science. The purpose of the Protein Refolding Screen Kit is to help simplify the process of identifying the buffer composition and method which is best suited for the refolding of any given protein.
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 give protein is a daunting challenge. For a 100 residue polypeptide there are 9e100 accessible confirmations. If each conformational search requires 10e-15 seconds to complete, it would take approximately 2.9 x 10e79 years to examine each possible configuration. This Levinthal paradox is resolved during protein folding by the progressive stabilization of intermediate states. Productive partially folded confirmations 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 significant 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 affecting 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 self-nucleating, 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 buffer exchange where the renaturation buffer is designed to minimize these offpathway products.
The folding of proteins in solution is affected 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 Clark (4). The first step to develop a method for refolding proteins purified from inclusion bodies is to determine the composition of the refolding solution. The Protein Refolding Kit contains 15 different buffer compositions which permit the rapid identification of the factors which are having a major effect 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 buffer with the refolding buffer including dilution, dialysis, diafiltration, gel filtration 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 buffer 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 buffer 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 final 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 final buffer (example: lysozyme) or by gradually removing the denaturant by dilution during dialysis (example: immunoglobulin G7). 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 buffer 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.