An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS.
Co-crystallization of membrane proteins with antibody fragments may emerge as a general tool to facilitate crystal growth and improve crystal quality. The bound antibody fragment enlarges the hydrophilic part of the mostly hydrophobic membrane protein, thereby increasing the interaction area for possible protein-protein contacts in the crystal. Additionally, it may restrain flexible parts or lock the membrane protein in a defined conformational state. For successful co-crystallization trials, the antibody fragments must be stable in detergents during 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 GOLD library (Morphosys, Munich, Germany). By combining the most stable and well expressed frameworks, V(H)3 and V(kappa)3, 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 pneumoniae. We describe efficient 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 recognize conformational epitopes with affinities in the low nanomolar range.
Röthlisberger
Daniela
D
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Pos
Klaas Martinus
KM
Plückthun
Andreas
A
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation
D001426:Bacterial Proteins; D002352:Carrier Proteins; C086246:CitS protein, bacteria; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D019151:Peptide Library