We want to regulate expressed proteins within cells at the post- translational level. Our approach is to provide an alternative to antisense technology. To accomplish the task we have molecularly re- engineered the DR1 or DR3 region of gamma globulin heavy chain (HC) gene fragment by randomization processes to produce a recombinatorial phage library of approximately 10(12) different immunoglobulin fragments having different antigen recognition sites. The library can be screened to find an individual clone that recognizes a given protein by fusing the recombinant to the M13p8. We have shown that we can isolate a newly engineered recombinant protein found within the large random library that binds specifically and tightly glucose-6-phosphate dehydrogenase (G6PD). In this particular case G6PD was chosen as a target because it is a constitutive enzyme and functions as an intermediate in the monophosphate shunt that produces reducing equivalents in the form of NADPH which is important for studies related to glutathione metabolism, as well as redox active drug metabolic studies. We had to remove the leader sequence of the HC gene cassette so that the newly expressed protein would not be excreted but rather would be expressed and retained within the cytoplasm. Additionally, methods for """"""""fishing out"""""""" the individual phage (s) that is (are) expressing the HC that binds G6PD and also inactivated G6PD had to be devised. Once we have shown that the paradigm works for G6PD, we will be able, by using the same library, to find proteins that may contribute to repair of or resistance to ionizing radiation or proteins that are within the apoptosis pathways. At a larger level, the methodology provides a new path to gene therapy for treatment of cancer or virally infected cells. The project requires knowledge and use of immunoglobulin gene structure, gene and protein fusion expression systems, site directed mutagenesis, polymerase chain reaction, nucleotide sequencing, plasmid and vector construction, protein screening, and other biologic techniques, as well as tissue culture.
