The bottom panel shows the key for clone locations around the master plates
The bottom panel shows the key for clone locations around the master plates. single variable domains binding to RAS and LMO2 oncogenic proteins. INTRODUCTION The velocity and versatility with which monoclonal antibodies (human or mouse) can be isolated has increased since the first K?hlerCMilstein description (1), partly due to phage display technologies that have rendered isolation of single-chain Fv (scFv, comprising linked VH and VL segments) (2) or single V domains achievable (3,4). The importance of high-quality reagents in a number of cell and molecular biology applications (5) necessitates the development of convenient technologies that can be applied in basic research laboratories as tools but also for clinical use where mouseChuman chimaeric monoclonal antibodies (6,7) or humanized versions (8) are making a major impact. A number of important laboratory techniques also require antibodies that can bind to Flopropione native, intracellular proteins, such as flow cytometric detection of intracellular proteins, using cell permeabilization (9), and immunoprecipitation-based methods including pull-downs, analysis of protein complexes and chromatin immunoprecipitation analysis. Single-domain antibody (Dab) Flopropione libraries have provided sources of antibody fragments that can be used as antibody reagents (10) or as Flopropione intracellular antibody fragments (11). Furthermore, single domains that bind to native proteins can be preferentially isolated using library screening in the yeast intracellular antibody capture (IAC) technology (12,13), because the target protein is expressed inside the cell within a normal cellular environment. In addition, the method can detect previously silent epitopes that may, for instance, fall within small clefts in antigen targets. Whilst the isolation of single-domain antibodies has been made simple by screening with phage display and yeast, obtaining compatible pairs of VH and VL segments that bind at the same epitopic region requires additional actions. Furthermore, scFv obtained by phage display do not usually provide complementary binders to a single epitopic region because of the way that this scFv are built from individual VH and VL segments. Ideally, engineering an scFv from single domains for binding native proteins could involve screening libraries of V regions to select a single domain of choice followed by a second screen that depends on the co-location of VH and VL around the antigen surface. We describe such a method (CatcherAb) for drawing together diverse single domains (VH and VL) into a high-affinity Fv format that can form the basis of a total antibody (which can have any tag required, be of any class required and of any species). The method involves the mandatory conversation of VH and VL domains in contact with native antigens as the basis for selection. We illustrate this with the selection and engineering of two unique Fv that specifically bind to their antigenic target (namely oncogenic RAS or LMO2) at the same epitopic location. The final scFvs have nanomolar affinity. MATERIALS AND METHODS Plasmids The yeast vectors, pBTM116-HRAS(G12V) and pVP16*, are explained in detail elsewhere (13). pBTM116-LMO2 was constructed by cloning the N-terminal truncated cDNA (14) HEY1 into EcoRI-SalI sites of pBTM116 vector. The pCatcher plasmid is usually described in detail in Supplementary Physique S1. The mammalian expression vectors, pM-HRAS(G12V) (Gal4-DBD fusion bait) and the pEFVP16 (VP16-AD fusion prey), are explained elsewhere (13). pM-LMO2 was constructed by cloning the N-terminal truncated cDNA into EcoRI-SalI sites of the pM vector. pEF/myc/nuc/VH was constructed by sub-cloning the anti-RAS VH#6 (13) or anti-LMO2 VH#576 (TT and THR, unpublished results) into the NcoI and NotI sites of pEF/myc/nuc (Invitrogen). All Flopropione constructs were sequenced to confirm in-frame fusion of the inserts with transmission peptide or fusion partners. Yeast single-domain VL library construction Single-domain human VL libraries were constructed in the yeast prey vector pVP16*. For the initial library construction for step-one screens (Physique 1A), a mixture of VL DNA fragments were PCR amplified from your human scFv phage libraries I or J (15) as template with the primers SfiVLF and pHENSeqR (all primer sequences used in PCR are shown in Supplementary Table S1). The PCR products were sub-cloned into the SfiI-NotI sites of pVP16*. The constructed VL libraries are composed of a single framework for Vk1 and diversified at two residues in CDR2 and five residues in CDR3.