Expression and purification of cancer-associated proteins are prerequisite steps necessary to study their functions, structures, and interactions. However, these steps are often challenging or even infeasible due to poor protein solubility. This project seeks to develop a tool, a set of entropic bristle-based AquoProtTM vectors, to dramatically increase the fraction of cancer-related proteins that can be expressed and purified in vitro, thus improving the success rate of functional analysis efforts for commercial and academic applications. Therefore, AquoProtTM benefit cancer research by enabling researchers to successfully purify and study many cancer-associated proteins that were not soluble with other technologies. This demand in novel solubiluization technologies is expected to grow in the market as proteomics projects proliferate, and as researchers are faced with the additional need to express recalcitrant proteins. The overall project aim is to develop a set of entropic bristle domain (EBD) fusion vectors, AquoProtTM kits that will be used to increase the solubility of many types of recombinant cancer-related proteins. The term """"""""entropic bristle"""""""" describes a highly flexible, non-aggregating polymer chain. The feasibility and effectiveness of the EBD fusions for solubilizing proteins previously characterized were successfully proven in Phase I extension research. Phase II is dedicated to the creation of the AquoProtTM vector, the expansion of the product line to develop a set of AquoProtTM vectors, the assembly of the prototype AquoProtTM kit, and the elaboration of the kit production cycle. The set of EDB-based AquoProtTM kits proposed here is a totally novel approach to attack the problem of cancer-related protein insolubility, whether it be a single protein or multiple targets. These solubility-enhancing kits will increase the utility of systems for cancer protein over-expression. It is necessary to emphasize that protein insolubility is a general and wide spread phenomenon. It slows down or makes completely infeasible studies on many proteins in pharmaceutical, agro-biochemical, biotechnological and academic laboratories and institutions. Therefore a set of vectors aimed at the enhancement of protein solubility will be of benefit for any researcher facing the insolubility problem. Public Health Relevance: Expression and purification of cancer-associated proteins are prerequisite steps necessary to study their functions, structures, and interactions. However, these steps are often challenging or even infeasible due to poor protein solubility. This project seeks to develop a tool, a set of entropic bristle-based AquoProtTM vectors, to dramatically increase the fraction of cancer-related proteins that can be expressed and purified in vitro, thus improving the success rate of functional analysis efforts for commercial and academic applications. Therefore, AquoProtTM benefit cancer research by enabling researchers to successfully purify and study many cancer-associated proteins that were not soluble with other technologies.
| Santner, Aaron A; Croy, Carrie H; Vasanwala, Farha H et al. (2012) Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression. Biochemistry 51:7250-62 |
