In various proteomics initiatives there is a significant effort to determine detailed molecular structures of the thousands of proteins and protein complexes that govern various cellular processes. Protein structural analysis by X-ray crystallography and NMR requires tens of milligrams of highly purified proteins. Over the past five years, the Protein Structure Initiative has been successful in developing high-throughput methods for structural determination of recombinant proteins, focusing on the so-called """"""""low-hanging fruit"""""""", i.e. proteins which are expressed at high levels as soluble, correctly folded species in E. coli, and which are relatively easily purified and crystallized. However, many targets important for human health, such as mammalian regulatory and membrane proteins, have thus far been left behind as """"""""high-hanging fruit"""""""" due to difficulties with one or more steps in the process. One major hurdle is the fact that many of these proteins are poorly expressed and/or undergo aberrant folding in E. coli, necessitating the use of eukaryotic systems, such as insect cells, as the expression host. The use of more complex expression systems introduces additional challenges for purification due to the higher proportion of non-target and interfering proteins in cellular extracts. In these cases the affinity purification schemes developed for bacterial-expressed proteins fail to produce the purity required for structural analysis, necessitating additional purification steps which are expensive and difficult to automate. To address this problem, we will test the concept of adapting a simulated moving bed (SMB) approach to the multi- milligram scale purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). In the SMB method, the solid phase moves in a countercurrent direction relative to the liquid flow in a continuous loop. Multiple chromatographic cells are arranged in a series with continuous input of feed and eluant streams and continuous output of raffinate and eluate streams. The most tightly bound species are released first, reducing retention time of bound species and minimizing peak dispersion. Elution of purified target species can be easily optimized by adjustment of buffer composition and flow parameters, such that the system continuously resolves the strongest binding species from weakly- and non-binding species. Historically, SMB chromatography has been almost exclusively applied to large-scale binary separations of small molecule isomers. We will first develop a prototype """"""""mini-SMB"""""""" device that overcomes previous mechanical barriers related to scaled-down SMB devices, and then we will test the device vs. standard methods in IMAC purification of three oligohistidine-tagged recombinant human kinases expressed in insect cells. Our project will determine if the inherent advantages of SMB chromatography can be successfully applied to IMAC purification of high-value recombinant proteins at the multi-milligram scale. ? This proposal describes a novel device and method that would facilitate the isolation of proteins of sufficient purity and quantity for reliable structural analysis. The determination of detailed atomic structures of the thousands of proteins that comprise human cells will greatly increase our understanding of the fundamental mechanisms of normal and disease states. Such information will lead to the discovery of new therapies and pharmaceuticals that can be precisely targeted to specific cellular pathways and/or proteins for more effective disease management. ? ? ? ? ?
