SBIR Phase I: Tool for cancer protein crystallization based on intrinsically disordered protein

This Small Business Innovation Research (SBIR) Phase I project will develop the use of unique artificial protein sequences, known as entropic bristles, to accelerate the study of protein structure by the method of X-ray crystallography. Many interesting proteins have proven to be difficult to crystallize, in part because they cannot be expressed and purified in a soluble, homogeneous form. A new approach is proposed to address this problem. Entropic bristles are highly charged, non-aggregating polypeptide sequences that lack a fixed tertiary structure. It has been found that fusing such a sequence to poorly soluble target proteins can dramatically improve their solubility. Bacterial expression vectors incorporating these sequences will be designed for the production of highly soluble fusion proteins that can be isolated at high concentrations. Crystal formation will then be initiated by proteolytically cleaving the bristle sequence under conditions suitable for crystallization of the isolated target protein. The Phase I objectives are (1) demonstrate that proteins previously crystallized by conventional means can also be crystallized by in situ proteolysis after being expressed as a fusion with an entropic bristle, (2) demonstrate that a protein that has not been previously crystallized can be expressed and crystallized using this method. The broader impact/commercial potential of this project will be to dramatically increase both the pace and the scope of protein structure determination. The method has two important advantages over existing techniques and products for protein solubilization: (1) the use of unstructured entropic bristles as fusion partners appears more generally effective, (2) it potentially provides a built-in mechanism for inducing crystallization. This new application of our entropic bristle technology will allow the three-dimensional structures of proteins to be determined more quickly and reliably. Knowledge of the structures of disease-related proteins is extremely valuable in designing drugs that target these proteins, and structure-based drug design has become one of the most common paradigms for discovering new drugs. The bacterial expression vectors and crystallization methods resulting from this work thus hold the promise of accelerating the field of structure-based drug design and facilitating discovery of new disease treatments. They will be of great interest both to pharmaceutical companies and many academic laboratories.