Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Abstract

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

New soluble angiopoietin analog of Hepta‐ANG1 prevents pathological vascular leakage

“…As the action(s) of endogenous Angpt1 is largely restricted to mediating pericyte‐endothelium communication, to create a systemic therapy to activate Tie2, we needed to consider how to overcome the “stickiness” or affinity of Angpt1 to rapidly bind ECM and become immobile. In addition, because the coiled‐coil structures mediate protein interaction and multimerization, the native Angpt 1 and its CCOD‐based mimetics also have tendency to aggregate during production and are unstable when stored in physiological buffer (Koh, 2013; Oh et al, 2015; Zalewski et al, 2017). To overcome these issues, we replaced the CCOD and linker sequence of Angpt1 with a “non‐sticky” 57 amino acid segment derived from serum complement C4‐binding protein α (C4BP) that naturally folds into a heptavalent scaffold, which allows the recombinant protein to circulate freely while maintaining potent Tie2 activating ability.…”