) Cell-free protein synthesis offers many advantages over in vivo expression for the synthesis of toxic, labile and labeled proteins and for the incorporation of unnatural amino acids. Analysis of in vivo protein production rates suggests great potential for rapid and efficient protein synthesis. However, current techniques are inefficient and unreliable. This proposed project will first develop robust methods for the batch synthesis of 100 nmole quantities of a variety of proteins. The methods will then be extended for efficient incorporation of labeled or unnatural proteins, either generally or site-specifically. This is the second revision of a proposal initially submitted 2/99. It has been extensively modified in response to reviewers' comments. In particular, the focus has been sharpened and the scope of work more clearly defined. In addition, the most serious concerns of the last reviewers have been addressed by recent results. In the last nine months, our approach has increased batch yields from about 100 to 500 pg/mI with PEP as the energy source. We have also demonstrated production of at least 77 nmoles of chioramphenicol acetyl transferase (CAT) in a single, 6-mI batch reaction. We have produced at least 80 nmoles in a single, 5-mI fed-batch reaction. This exciting progress has resulted from a fundamental analysis of reaction stoichiometry followed by innovative approaches to new technique development. Two new ATP regeneration systems have been introduced, the importance of phosphate accumulation has been recognized, and key amino acid limitations have been identified. Work is now proposed to modify the cell extract for removal of deleterious enzymes, to provide for more consistent cell extract preparation, to develop more efficient synthesis techniques, and to increase productivity by condensing the cell extract. We will then apply these improved methods to the introduction of labeled and unnatural amino acids. The work will be conducted with six different proteins representing a variety of biochemical characteristics and species of origin. When successful, this project will provide exciting new technology for the efficient synthesis and modification of a wide variety of proteins.
