Using molecular biology to bestow a characteristic on a product that permits its selective removal from a multicomponent cellular extract can be of immense benefit in the downstream processing of the products of biotechnology, including enzymes, hormones and antibodies. Theory and past results both indicate that the addition of charged tails to proteins can aid the selectivity of separation methods. This proposal seeks to fuse polypeptide tails to proteins in order to enhance the recovery of proteins. Beta-galactosidase has been chosen as a model protein to carry out this proposal. The work plan includes the following: 1. Design of the polypeptide tail. 2. Synthesis of the gene segment for the tail. 3. Cloning this segment into the beta-galactosidase gene. 4. Expression of the gene in E. coli. 5. Assessment of structural and/or activity changes in the target protein. 6. Recovery of the fused protein by precipitation and two-phase aqueous extraction. This grant application is an excellent example of the type of cross-disciplinary proposal we wish to see in the Engineering Directorate's Biotechnology Program. The Principal Investigators are considered to be well qualified to carry out the proposed research.
