An antibody library for stabilizing and crystallizing membrane proteins ^ selecting binders to the citrate carrier CitS

Abstract Co-crystallization of membrane proteins with anti-body fragments may emerge as a general tool to facilitate crys-tal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydropho-bic membrane protein, thereby increasing the interaction area for possible protein^protein contacts in the crystal. Additionally, it may restrain £exible parts or lock the membrane protein in a de¢ned conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents dur-ing the extended period of crystal growth and must be easily produced in amounts necessary for crystallography. Therefore, we constructed a library of antibody Fab fragments from a framework subset of the HuCAL GOLD0 library (Morphosys, Munich, Germany). By combining the most stable and well ex-pressed frameworks, VH3 and VU3, with the further stabilizing constant domains, a Fab library with the desired properties was obtained in a standard phage display format. As a proof of principle, we selected binders with phage display against the detergent-solubilized citrate transporter CitS of Klebsiella pneu-moniae. We describe e⁄cient methods for the immobilization of the membrane protein during selection, for ELISA screening, and for BIAcore evaluation. We demonstrate that the selected Fab fragments form stable complexes with native CitS and rec-ognize conformational epitopes with a⁄nities in the low nano-molar range