Tuesday, November 9, 2010: 8:30 AM
255 A Room (Salt Palace Convention Center)
Obtaining high-quality protein crystals remains the major bottleneck for structure determination, particularly in the case of membrane proteins. Diverse successful strategies exist to generate crystallizable proteins, however no generalized approach has been developed. One emerging method involves co-crystallization with a protein-specific antibody fragment, and is primarily used for membrane proteins. We report the development of a potentially generalized approach utilizing recombinant scFv antibodies capable of binding specific peptides that can be introduced into flexible loops or at the c-terminal end of client proteins. The scFvs were derived from a monoclonal anti-His tag antibody 3D5 (Kaufman) with a combination of site-directed and random mutagenesis to improve the affinity, solubility and expression of the scFv in E. Coli bacteria. The chaperone variants were screened for affinity to various peptide tags (EYMPME and hexahistidine) and characterized using SPR, ELISA experiments and gel filtration to examine their ability to complex with tagged proteins (~25-800nM affinity). Preliminary crystallization trials show the hyper-crystallizabiliity potential of these antibodies (structures solved at ~3Å). The ability for the recombinant proteins to target specific peptide tags that can be attached to any protein of interest offers a promising new path to production of high quality crystals by utilizing the hyper-crystallizable antibody for use as a crystallization scaffold.