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Abstract
Co-crystallization recombinant antibody fragments (crFabs) are antibody fragments derived from a parent antibody fragment that can be used to assemble a 3- dimensional matrix (crystal). Our hypotheses were the following: 1. A molecule of known structure can be modified by genetic recombination to draw another macromolecule along with it into a crystal matrix. 2. The unknown structure can be determined with the aid of the known component. We have the ability to determine macromolecular structures rapidly. A ratelimiting step is frequently at the crystal formation stage. Typically, only about 20% of macromolecules screened with a large variety of reagents by any available crystallization strategy produce usable crystals. With the development of crFab-based crystallization, those odds may be improved considerably. Four Fab mutants of a mouse recombinant antibody fragment were tested for their suitability as co-crystallization reagents. The first mutant had a 6-His purification tag on the C-terminus of the heavy chain of the Fab (Fab2). Since Fab2 did not crystallize, the second mutant was modified to have four vicinal histidines, two on each chain. It was discovered that this mutant had a leucine residue inserted into a beta-pleated sheet when compared with mFab25.3. Fab4 was then constructed by a deletion mutation of that residue. Finally a heavy chain (N57.K) mutation was created to engineer a stronger salt bridge between the Fab molecules as they packed into the crystal structure (Fab5). Mutant rFab4 proved to be the best mutant for co-crystallization studies based on its yield and crystallization properties. By multi-site directed mutagenesis of the third hypervariable regions of the light and heavy chains, a recombinant Fab antibody fragment library was built with a nominal diversity of 1.16 x 107 colony forming units. This library was shown to produce rFabs that could recognize seven out of seven proteins tested and has the potential for being used for the co-crystallization of proteins. INDEX WORDS: Antibody fragment library, Multi-site directed mutagenesis,