Cell-Free Protein Synthesis for High-Through-Put Proteomics

73169-The production of proteins from cloned DNA sequences is an important process for functional genomic studies and structural analysis, as well as many other research applications, including pharmaceutical drug discovery. This project will develop in vitro protein synthesis reagents and instrumentation that will produce highly active, robust protein using a universally applicable protocol. The system will generate 100 milligrams or more of active protein, allow it to synthesize multiple proteins in one reaction, and allow the process to begin with PCR (polymerase chain reaction) amplified DNA. The system will provide a tremendous advantage in simplicity and cost over in vivo protein expression methods in that it simplifies or eliminates vector construction, cell transfection, protein induction, and uncertainties of host cell synthesis. Phase I demonstrated that enhanced S30 wheat germ lysate could be made, that it could generate up to 30 milligrams of protein in one reaction, that the protein it generated was enzymatically active, and that the process could begin with either plasmid or PCR-generated template DNA. Seven different proteins of varying sizes were synthesized from bacterial and mammalian origin. The synthesized protein was successfully purified from the reaction mixture using a simple, one-step affinity purification method. Phase II will improve the system to allow for: (1) the generation of 100 milligrams or more of active protein, synthesis of multiple proteins in one reaction; (2) the process to begin with PCR amplified DNA; (3) affinity purification; and (4) the sale of these products at affordable prices, thus making the methodology available to any laboratory. Commercial Applications and Other Benefits as described by awardee: The technology should generate several products that can be sold directly to the 50,000 or more laboratories throughout the world that perform genomic and proteomic research. We project that purified protein can be synthesized by these products for less then $11 per milligram. In addition, a simplified instrument can be developed and marketed that will automate the incubation and substrate addition steps of the procedure. The instrument, which should cost less then $2,000, can be marketed with the reagent kits. These products will save countless man hours of time, improve the outcome of experiments, and represent a tremendous cost saving to laboratories.