Abstract
Determination of crystal structures of membrane proteins is often limited by difficulties obtaining crystals diffracting to high resolution. Co-crystallization with Fab fragments of monoclonal antibodies has been reported to improve diffraction of membrane proteins crystals. However, it is not simple to generate useful monoclonal antibodies for membrane protein crystallography. In this report, we present an optimized process for efficient screening from immunization to final validation of monoclonal antibody for membrane protein crystallography.
Highlights
Since the first high-resolution structure of a membrane protein, the photosynthetic reaction center, was solved in 1985 [1], more than 290 unique structures of membrane proteins have been reported
Crystallization of the membrane protein complexed with Fab or Fv fragments derived from monoclonal antibodies has, in a few cases, allowed new structures to be solved for ion channels, G-protein coupled receptors, and membrane transporters [2,3,4,5,6,7,8]
Screening of antibodies against discontinuous structural epitopes is typically performed using the experimental criteria of positive ELISA (Enzyme-linked immunosorbent assay) on membrane proteins in native conformations but negative western on SDS-denatured proteins [9,10]
Summary
Since the first high-resolution structure of a membrane protein, the photosynthetic reaction center, was solved in 1985 [1], more than 290 unique structures of membrane proteins have been reported (database for membrane proteins of known 3D structure, http://blanco.biomol. uci.edu/Membrane_Proteins_xtal.html). Crystallization of the membrane protein complexed with Fab or Fv fragments derived from monoclonal antibodies has, in a few cases, allowed new structures to be solved for ion channels, G-protein coupled receptors, and membrane transporters [2,3,4,5,6,7,8]. Screening of antibodies against discontinuous structural epitopes is typically performed using the experimental criteria of positive ELISA (Enzyme-linked immunosorbent assay) on membrane proteins in native conformations but negative western (or dot-blot) on SDS-denatured proteins [9,10]. Recent developments in phage and ribosomal display have resulted in faster screening procedures for antibody fragments and other crystallographic chaperones [12,13]; they both involve proprietary reagents not yet commercially available
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