The expression of recombinant proteins in bacteria occasionally results in the improper folding of the expressed proteins. Since proper folding is required not only for a protein’s activity but also for solubility, misfolded proteins frequently aggregate in insoluble complexes called inclusion bodies. Although inclusion bodies can simplify the purification of the recombinant protein, it is only helpful if active protein can be recovered from the insoluble mass.
With something as complex as the folding of a polypeptide chain into a fully active protein, it is no surprise that a plethora of protocols can be found in the literature. However, since the in vitro environment required to refold a particular protein cannot be predicted a priori, deciphering the optimal conditions is usually by trial and error and, hence, can be very time consuming and expensive. Fortunately, several vendors now offer kits to streamline the process. After reviewing kit components and instructions from several vendors, I decided to try the Pro-Matrix™ Protein Refolding Kit from Pierce (Rockford, IL; cat. no. 89867).
From reading the package insert included with the Pro-Matrix™ Protein Refolding Kit, it is apparent that much thought went into the design of the kit. The kit permits the testing of numerous combinations of conditions that have been found to be important in refolding of proteins in general; the hope being that at least one condition will result in the proper refolding of your protein of interest. The kit includes 9 different refolding buffers as well as several additives, either as solutions or dry powders, that can be added to the refolding buffers as needed. The buffers and additives facilitate the construction of a matrix of conditions which vary according to denaturant strength, redox environment, ionic strength, and divalent cation concentration. Other conditions such as protein concentration, pH, and temperature can also be varied. Significantly, the Pierce matrix is designed to screen through the buffer conditions that have proven to be the most broadly useful for many proteins. While it is entirely possible that the initial matrix will suffice to identify adequate refolding conditions, the various additives allow other conditions to be tested for further optimization.
I used the Pierce refolding kit in an attempt to salvage several milligrams of recombinant human cyclophilin A (CypA) that I had purified from E. coli. Although the resulting CypA had the expected molecular mass by MALDI, it failed to bind the well-known ligand cyclosporin A. Hence, it appeared to be improperly folded. After denaturing about 3 mg of CypA overnight in 8 M guanidinium HCL, 50 mM Tris-HCL, pH 8, 10 mM DTT, I first performed a spin-column buffer exchange to remove the DTT and then divided the protein among the 9 refolding buffers to which I had added EDTA, DTT, and different ratios of oxidized and reduced glutathione. The refolding was allowed to proceed overnight at 4°C.
The refolded proteins were then evaluated by measuring their circular dichroism (CD). This was accomplished my measuring the CD of the individual refolding buffers with and without CypA, and then subtracting the CD of the buffers alone from the protein-containing buffers. The results are shown below.
The heavier red line is the CD spectrum for undenatured, i.e. misfolded, CypA. Although we have yet to complete functional testing of the recombinant protein, it is encouraging that at least 5 of the refolding conditions resulted in proteins with CD spectra clearly different from that of the misfolded protein. This implies that the proteins have, indeed, been denatured and refolded in a manner that is distinct from the original protein.
In summary, the Pro-Matrix™ Protein Refolding Kit from Pierce is a convenient, flexible, and economical means of testing various proven protein refolding conditions. The kit contains sufficient reagents to perform 100 refolding experiments of 1 ml volume and costs around $280. Considering the time it would take to accumulate this array of reagents and assemble them into different refolding buffers, the kit is quite a bargain.
Michael Campa, PhD
Assoc. Research Professor of Radiology
Duke University Medical Center
Radiology