A Rapid Method for Refolding Cell Surface Receptors and Ligands.

Cell surface immunoreceptors, ligands and viral decoys (receptors and ligands) belong to a major class of membrane receptors that play vital roles in a variety of biological processes. These include molecular recognition, viral infection, and immune defense. The successful expression and purification of these host and microbial surface proteins in active forms are critical for biochemical, structural, and functional studies of receptor-ligand molecular recognition in the context of host-pathogen interactions. Among the variety of challenges associated with the study of this class of molecules, obtaining purified functional receptor and ligand proteins is often a long and labor-intensive process. Because of the limitations in obtaining targeted cell surface molecules from native tissues and cells expressing the proteins, recombinant forms of receptor and ligand proteins are often the preferred choice for investigations. Among the heterologous systems used to express target proteins (integral membrane proteins excluded), bacteria hosts represent the most economical system in terms of associated costs, speed, and ease of use. However, cell surface proteins partition to the inclusion body (IB) fractions in majority cases when overexpressed in E. coli. This makes inclusion body-based receptor/ligand refolding often a required step to obtain functionally active cell surface proteins (for reviews on general methods in inclusion body-based protein refolding, see references1,2,3). Multiple cases of refolding-based receptor production have been reported4,5,6,7,8,9. Traditional chaotrope-based refolding techniques, however, require purified inclusion bodies of the target proteins of interest, which in turn require extensive washing steps (traditional solid-phase or centrifugation-based) that can take multiple days (Fig. 1A and Supplemental Figure S1). Typical refolding approaches involve either step-wise gradient dilution to dialyze away high concentrations of chaotrope agents such as urea or drop-wise dilution of denatured inclusion bodies directly into a refolding buffer. The entire process of refolding for cell surface proteins can span from one to over two weeks (Fig. 1A and Supplemental Figure S1)4,10, yet the success rates of these refolding approaches are usually low11. Lengthy turn-around times, labor-intensive processes, and low chances of successful refolding make these refolding approaches daunting. Therefore, investigators need to expend significant amounts of time to find optimal conditions in a trial-and-error fashion. During these lengthy processes, the amount of chemical and biochemical reagents consumed to aid the refolding process (such as large quantities of the chaotrope reagents urea and guanidine hydrochloride (GuHCl), protein aggregation inhibiting-reagents such as arginine, reducing and oxidizing reagents, etc.) is considerable. Despite all the technical challenges, these approaches are routinely used to generate many cell surface receptor and ligand proteins with intact native structures and active functions3,4,5,6,7,8,9. On-column refolding of proteins (including receptor fragments or domains) using immobilized metal ion affinity chromatography (IMAC) has been reported in several studies6,7,8. These studies were typically using highly purified IBs before on-column refolding and each refolding was usually optimized for a single target. Figure 1 Schematic representations of time required for refolding in different methods, illustration of targeted cell surface proteins sequence domains, and protein structures and models. One approach to cope with technical challenges in protein refolding is to apply high throughput screening. Developments of such refolding screenings have been reported, based on analytical detection of reverse phase-fast protein liquid chromatography (rp-FPLC) or differential scanning fluorimetry (DSF)12,13. These studies focused on expanding the capability to accommodate large numbers of proteins. For example: establishing a 96-well-plate format so that refolding conditions may be optimized rapidly. We sought to develop a rapid and general method for refolding cell surface receptors and ligands that will fill a much needed technology gap for an important class of biomacromolecules in structural biology, functional genomics, and chemical biology applications. Built upon works from literature and our systematic efforts to refold a variety of cell surface immuno- and viral receptors and ligands, we report here a rapid and less expensive method that successfully refolded membrane-associated receptors and ligands with different classes of structures and functions.