W0042

Crystallization of Macromolecules Using Self-Assembling Matrices and Phage Display. Cory Momany, Departments of Botany and Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30604 USA

Crystallizing macromolecules and obtaining phases are the rate limiting steps in macromolecular structure determinations. One solution to both problems is to use self-assembling matrices (SAMs) that carry molecules into their lattices- akin to the host/guest situation in small molecule crystallography. What is required is a macromolecule that forms a large lattice, but recognizes a variety of guests. The antibody Mab25.3 against the HIV capsid protein when re-engineered and expressed as a Fab on the surface of filamentous phage for selection, satisfies these criteria.

Heavy and light chain cDNA were obtained by RT-PCR of mRNA from a hybridoma producing the Mab25.3 antibody. The cDNA were cloned into the Phage Display vector pComb3H, the gene III protein removed, and a poly-histidine purification tag added. Expression was then optimized by mutating rare codons and by lowering the induction growth temperature in the presence of Mg²⁺. Currently, up to 5 mg of purified Fab can be obtained from the periplasmic fraction per liter of cells using metal-chelate affinity chromatography. Significant amounts of Fab are also excreted into the media, but the his-tag appears to be removed. The expression levels of the Fab are still inadequate for use in crystallography Thus, improving the expression is a high priority.

Crystallization trials are now in progress using the recombinant Fab. Once satisfactory expression of Fab that crystallizes is obtained, a combinatorial library will be introduced into the CDR loops of the Fab. Ultimately, Phage Display selection using this library should allow many proteins to be simultaneously recognized and crystallized. Since the lattice is known, molecular replacement can be used to rapidly solve the structures. The combination of fast crystallizations and quick structure determinations using a SAM-based approach means that X-ray crystallography can make significant contributions to genome projects.

Funding by the National Science Foundation is gratefully acknowledged.