Protein Refolding Kit

Introduction

Reasons 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, misfolds1.

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, and 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.


Principles of the Kit

The information for protein folding is coded in the linear sequence of the polypeptide2. 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 9100 accessible confirmations. If each conformational search requires 10-15 seconds to complete it would take approximately 2.9 x 1079 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 reviewed3,4,5. 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 Clark4. Thus, 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 Screen 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 II6. 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.

Components

Buffer Preparation:
Before using the Buffers add 1 ml DTT, GSH or GSSH solutions to 950 ml of the respective buffer as follows:

Protocols

Protocol 1:   Screening for the basic buffer composition
Because there are a myriad of chemical and physical conditions which can affect protein refolding, the systematic survey of each factor would be a time consuming process. The screen employed by this kit is based on a fractional factorial experimental design. This allows the researcher to determine the critical factors affecting protein refolding and thus to quickly define a suitable refolding regime.
 

Example analysis table used to determine the critical factors to refolding. (An Excel spreadsheet file is available for download at www.athenaes.com/TechnicalRef.htm)

Protocol 2:   Optimization of the buffer composition
Once the critical parameters have been identified, the refolding conditions should be optimized. The extent to which the optimal conditions must be defined depends on the intended use of the protein and whether or not additional purification steps are needed. The following is a general scheme for optimizing the refolding procedures.
 

Protocol 3:   Purification of Inclusion Bodies
There are a myriad of approaches for purifying inclusion bodies. The most common technique for bench-scale applications is centrifugation, with diafiltration and continuous flow centrifugation being used for commercial-scale operations. It is most often best to purify the inclusion bodies as insoluble products and the dissolve them in denaturant before refolding. This will remove unwanted contaminants, especially proteases. Once the inclusion bodies are relatively pure, they are solublized with 6 M guanidine HCl or 8 M Urea. The solubilization is protein-dependent and the conditions needed with regard to the concentration of denaturant, ratio of denaturant to protein, pH, ionic strength, time of exposure to denaturant, temperature, redox agents or derivatization of thiol groups should be determined empirically. The method below is a general scheme which will work for most proteins. Alternative approaches and more extensive discussions on inclusion body purification can be found in the literature.3,4,5,11
 

Supplemental Protocol 1:   Methods for increasing soluble protein accumulation
Before embarking on experiments to define a protein refolding regime, it is advisable to first determine whether or not the target protein can be recovered in a soluble and therefore presumably native state. Two relatively simple and quick tests are recommended; a media screen and induction of chaperone proteins. It is known that medium composition can effect the accumulation of recombinant proteins. Likewise, the relative fraction of soluble protein of an otherwise insoluble product is affected by an as yet undetermined mechanism. To determine whether medium composition affects a particular protein, several media formulations should be screened for accumulation of the target protein in soluble extracts.

The following protocol is for use with Athena’s Media Optimization Kit™ or APF Media Optimization Kit™. The method can be adopted for use with any set of media formulations desired.

Prepare the media as per the kit instructions
 

Perform the media screen as follows:

Supplemental Protocol 2:  Increased soluble protein accumulation using chaperone induction

Other factors which can increase the accumulation of soluble protein are chaperonin proteins. Chaperones are a class of proteins found in all organisms which play a role in folding of protein or the refolding of mis-folded proteins. Several studies have shown that the co-expression of selected chaperones increases the accumulation of soluble protein during hyper-expression1. However, in vitro studies have found that not all proteins are acted on by chaperones uniformly12. In other words, while the accumulation of soluble forms of some proteins can be increased by chaperones, other proteins are unaffected. At the present time no classification scheme is available to allow one to predict which proteins are likely to be acted on by a give chaperone or set of chaperonins. Therefore, trial and error testing would be needed to identify a suitable chaperone(s) for a given protein. Complicating this is that not all chaperone proteins are available in sufficient quantities for refolding work. As an alternative, Athena’s scientists have developed a medium additive, Augmedium™, which induces the expression of chaperones. Rather than co-expression one specific chaperone, Augmedium™, causes a sublethal chemical and oxidative stress which results in the expression of a range of chaperone proteins. In this way, prior knowledge of which family of chaperones that act on the target protein is not needed.

References