In the second grant we demonstrated that novel low molecular weight (MW) displacers can be successfully employed for high resolution/high-throughput protein purifications in ion exchange systems. We demonstrated that low molecular weight displacers can also be used for oligonucleotide purification and that the stationary phase has a significant effect on the efficacy of various low molecular weight displacers. Preliminary results indicate that high-throughput screening can be successfully employed to evaluate a wide variety of displacer chemistries. Further results from our laboratory indicate that this data can be employed in concert with novel quantitative structure efficacy relationship models to both predict and interpret displacer efficacy. Finally, preliminary results in our laboratory indicate that chemically selective displacers can be identified which can dramatically increase the selectivity of ion exchange systems. In the next phase of this project we will incorporate these new lines of research into our ongoing investigation into the rational design of displacers for the purification of biomolecules. We will incorporate the tools of high-throughput screening and synthesis, and quantitative structure activity relationship models into the research and we will address several critical issues related to the design of both high affinity and chemically selective low MW displacers for proteins and oligonucleotides in ion exchange systems. The next phase of the project will include high throughput screening, quantitative structure activity relationship models, displacer synthesis, chemoenzymatic synthesis of displacer libraries, the development of high affinity displacers for cation and anion exchange systems, development of selective displacers, and the application of these displacers for purification of biomolecules from complex mixtures. The synthetic work will include structural variants of low MW displacers identified in the second grant as well as variants of other promising candidates recently identified in our laboratory. The displacers developed in this research will be employed in a detailed investigation of the interplay between displacer chemistry and its ability to act as a high affinity or selective displacers for various classes of separation problems. This research will evaluate several hypotheses related to displacer design. The low MW displacers developed in this research will be employed for the purification of proteins and oligonucleotides from complex biological mixtures. Finally, these high affinity displacers will be employed to develop a generic displacement technique for purification of a range of proteins with possible applications to proteomics. The proposed research will provide significant insight into the design of efficient low MW displacers for protein and oligonucleotide purification and will enable more efficient purification of a wide range of biopharmaceuticals. Furthermore, this work will facilitate the use of this high resolution/high throughput technique by medical researchers at the bench scale for the rapid purification of novel biomolecules from dilute complex mixtures. ? ? ?
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