Crystallization of Antibodies and Antibody-Antigen Complexes

Abstract

Although many antibodies have been crystallized, the number of structures determined in both their complexed and unliganded forms remains relatively small. With the recent improvements in the use of molecular replacement (MR), the structure determination of Fabs and Fab-complex structures can proceed more rapidly, but crystallization often remains a major obstacle. Substantial improvements in methodologies have helped with the success rate in the crystallizations of Fabs and Fab-antigen complexes that are beyond previous expectations. Crystallization and structure determination have been directed mainly toward Fab fragments. The reason for this choice remains linked both to the ease with which the structure of Fabs can be determined and to the difficulties that have been presented by the crystallization of whole immunoglobulins. Such difficulty is currently believed to be due to flexibility or conformational heterogeneity of the IgG as well as the added heterogeneity from the glycosylation of the Fc fragment. Fabs share some of the same problems mainly because of the degree of heterogeneity that is the result of the proteolytic cleavage used to fragment the immunoglobulins, of the flexibility in elbow regions, and in some cases of glycosylatlon. A systematic approach to the cleavage, purification, and analysis of the resultant product can yield inmunoglobulin fragments amenable to crystallization. A rational screening of crystallization conditions with extensive use of seeding can In most cases enable progress from small microcrystalline aggregates to large X-ray-quality crystals. Such methodologies have become so effective that Fabs are now being used as tools to aid in the crystallization of other molecules that have been found difficult to crystallize by themselves.