Proteases and tags for protein purification and analysis

DESCRIPTION (provided by applicant): The long-term objective is to commercialize a system for rapid purification of proteins from cloned genes. The system should be suitable for both high throughput, parallel purification of proteins on the laboratory scale, as well as process-scale purification of pharmaceutical proteins. This technology was developed as a result of NIH-funded studies on the prodomain- mediated folding reaction of the Bacillus protease subtilisin. The two fundamental components of this system are: 1) A highly engineered protease (psub) which hydrolyzes specific substrates in response to a fluoride trigger; 2) The high affinity interaction between psub and an engineered version of its prodomain (protag). This components are combined to create a purification system in which immobilized psub is used both as the binding molecule for affinity purification of protagged fusion proteins and as the processing protease for protag removal. The four experimental aims are: 1) Engineer site-directed immobilization of psub; 2) Optimize large scale production and purification of psub; 3) Create and test high-throughput methods for protein purification, quantitation and analysis; 4) Identify second generation psubs with refined chemical triggers. Recombinant proteins are frequently fused with other proteins or peptides to facilitate expression and purification. The tags provide a temporary hook for affinity purification, but ultimately must be processed by a site-specific protease. Tag removal, however, is frequently inefficient and sometimes problematic. The technical innovation of the system is the integration of tag removal into the purification process. This provides simplicity and efficiency that isn't available in any other system. The technology should benefit anyone purifying proteins but the impact on structural genomics efforts which rely on parallel processing of samples should be particularly great. The technology should eventually benefit process scale purification of pharmaceutical proteins. The ability to rapidly produce large quantities of therapeutic proteins could be critical for responding to both naturally-emergent and intentionally-introduced pathogens. The technology being developed in this project should benefit anyone purifying proteins but the impact on structural genomics efforts which rely on high throughput, parallel processing of samples should be particularly great. The technology should eventually benefit large-scale purification of pharmaceutical proteins. The ability to rapidly produce large quantities of therapeutic proteins could be critical for responding to both naturally-emergent and intentionally-introduced pathogens.