(571az) Engineering Hyper-Crystallizable Single Chain Antibodies for Crystallization Scaffold

Crystallization of protein has been limited by the expression and purification of the protein as well as the identification of conditions that promote crystal growth. Using protein engineering, we allow the protein to become the variable in crystallography thereby offering a promising new pathway to the production of high-quality crystals. Genes for a hyper-crystallizable single chain antibody (scFv/scAb) with specificity for a peptide tag (EE tag; amino acid sequence EYMPME), were cloned and expressed in E. coli bacteria. CDRs with this EE-tag specificity have also been grafted onto frameworks of previously crystallized antibodies and randomly evolved. The antibodies were engineered with the capability of crystallizing in multiple space groups and lattice structures and intended for the development of a crystallization scaffold and intended for the development of a crystallization scaffold. Variants of the antibodies selected by phage display had specific EE-tag binding affinity, soluble expression and high stability. Directed evolution was performed to preserve amino acids that were significant in maintaining crystal lattice contacts. CCP4 program suite for protein crystallography was used to derive locations for directed evolution. These locations were selected based on surface area energetic and crystallographic work on the original antibodies from which the grafts derived their framework. The engineered single chain antibodies produced were tested for their ability to crystallize independently using the Phoenix robot at the University of Texas. The generation of a hypercrystallizable antibody will allow for a readily formed complex with any second protein of interest by simply attaching the EE-tag. Its use can then be extended to create scaffolds aiding the crystallization of proteins that are otherwise difficult to crystallize, producing a general engineering approach to creating crystalline proteins.