After panning, a first screening is conducted by one-point ELISAs with the supernatants of independent selected clones using unpurified Fab from the final round of panning. resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF–ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12C419 nM and performed unfavorable stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the MDL 28170 Fabs have overlapping or impartial epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation Rabbit polyclonal to ANTXR1 including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins. Introduction HIV, like other viruses, is an obligate intracellular parasite and requires host cellular machinery at all stages of its life cycle to replicate and counteract the effect of the host immune system. During contamination, viruses manipulate the cellular mechanisms of host organisms via pathogen-host interactions to take advantage of the capabilities of host cells, leading to viral replication. Understanding the structure and function of human host factors and host-virus interactions is essential for a complete knowledge of the viral contamination and the identification of new targets for therapeutic intervention. However, structural studies of HIV-host complexes have been hindered due to the high degree of flexibility of the complexes, their dynamic nature and the resulting reversibility of complex formation. To assist functional studies, X-ray and cryogenic electron microscopy (EM) structural studies of difficult to study proteins and protein complexes we MDL 28170 have developed conformation-specific recombinant antibodies for protein and complex stabilization, capture, and labelling [1C4]. Fabs (fragments antigen binding) have been used to facilitate crystal packing of soluble and integral membrane proteins by binding specific epitopes resulting in stabilization of desired conformations [5C7]. Fabs have also been used in unfavorable stain EM to label domains within a complex [8, 9] and to increase the size of the target protein and provide a fiducial mark with recognizable features for image alignment and better visualization in cryo-EM [3]. Highly specific monoclonal antibodies are usually generated through hybridoma technology or by biopanning with antibody libraries with the target of interest. Non-antigenic sequences in a protein or the need for three-dimensional epitopes can make antigens hybridoma-refractory and particularly challenging targets for monoclonal antibody identification since the complexes are unstable and do not maintain their three-dimensional structure during the immunization process. In the case of HIV, three critical dynamic protein assemblies are VCBC [10]; the ESCRT-I [11]; and the clathrin adaptor protein complex 2 (AP2) [12] (summarized in Fig 1). The accessory viral protein Vif (Viral infectivity factor) induces the ubiquitination and degradation of the APOBEC3 (Apolipoprotein B Editing Complex) family restriction factors by binding to the ubiquitin ligase adaptors Elongin B and C (ELOB/C), core binding factor beta (CBF-) stabilizes the formation of this complex. The HIV structural protein Gag recruits the cellular endosomal sorting complexes required for transport (ESCRT) machinery to facilitate viral budding. The viral accessory protein Nef (unfavorable factor) induces the degradation of the CD4 receptor in the lysosome by binding to the cytosolic tail of CD4 and AP2. To further study these complexes, we have identified Fabs from a na?ve human antibody library [13] that recognize conformational epitopes around the complexes with high specificity and high affinity to their cognate antigen. Open in a separate window Fig 1 Three protein complexes essential for HIV replication targeted with recombinant antibodies.The antibodies described here are shown in red. Here we report well-characterized antibody fragments that recognize HIV-host protein complexes that can aid in obtaining high resolution molecular structures and functional characterization of these complexes. To identify these recombinant antibody reagents, we developed a screening pipeline using quantitative ELISAs and unfavorable stain EM (nsEM) to rapidly and MDL 28170 cost effectively identify and test binders, prior to large scale expression and purification of the complexes. The.