Specific buffers were used for each target membrane protein and are referred to as Tg1 cells (OD600= 0.5C0.8). (ELISA) and unpurified Fabs. This procedure greatly speeds the prioritization of candidate binders to membrane proteins and will aid in subsequent structure determinations. when the cells are infected with helper phage, which harbors the full phage genome necessary for completing the phage life cycle. Thus the antibody fragment is presented on the phage surface while its encoding gene resides within the phage particle. Antibody presenting phage particles are subject to a procedure called biopanning or panning, which refers to repeated cycles of binding between phage and immobilized antigen, washing away unbound phage, eluting bound phage, infecting with the eluted phage and propagating the eluted phage (Fig. 1). Thus, the phage particles that display antigen-binding antibody fragments become enriched. Relevant phagemids can be extracted from the cells infected with the eluted phage. Open in a separate window Fig. 1 A panning scheme for membrane proteinsIndirect immobilization is applied where biotinylated membrane proteins are captured by streptavidin magnetic beads. Panning consists of 1) capturing membrane proteins, 2) binding of Fab phage and captured membrane proteins, 3) washing away unbound phage, 4) eluting bound phage, 5) infecting Tg1 cells with the eluted phage and 6) propagating phage for the subsequent round of panning. Three rounds of panning are performed. The 3rd round selected Fabs are subject to ELISA analysis to identify Fabs that bind to target membrane proteins. In comparison to Diphenyleneiodonium chloride hybridoma antibody production, which can take several months, phage display antibodies can be obtained in a few weeks and provide a stable, renewable source of antibodies. selection of phage display does not rely on antigen immunogenicity, lack of which is a major limitation of hybridoma antibody production, and thus extends a range of target antigens . TNFRSF10D Diphenyleneiodonium chloride The antigens are presented in their native state, allowing for the recognition of three-dimensional epitopes by the antibodies. Manipulation of selection conditions also facilitates generation of phage display antibodies with desired specificity. Additionally, phage Diphenyleneiodonium chloride display is a relatively straightforward and cost effective technique to set up. Selected antibody fragments can be easily produced in large quantities. Thus, phage display biopanning is a powerful method for identifying antibody fragments used for crystallographic studies of membrane proteins. Despite these advantages, biopanning against membrane proteins brings forth Diphenyleneiodonium chloride some challenges. Antigens need to be soluble throughout the panning process to ensure selection of antibodies recognizing native forms. This can be problematic for some membrane proteins because they are typically less stable than soluble proteins. Nonetheless, membrane protein aggregation can be minimized by proper stabilizing additives . Even well solubilized and stable membrane proteins have reduced solvent exposed surface area compared to soluble proteins of equal size due to micelle formation around the transmembrane segment. This reduces the potential antibody binding surface, reducing the chance of selecting binding partners for highly compact membrane proteins with little solvent exposed surface area. Nevertheless, numerous antibodies against membrane proteins have been successfully generated by phage display biopanning [6, 8, 14]. Successful identification of a Fab that stabilizes a membrane protein requires a strategy to rank the numerous candidates resulting from a biopanning experiment. A precise determination of the binding energy for each candidate is preferred, but quickly becomes untenable when working with hundreds of putative binders using current technologies. For crystallographic studies, determining the binding energy for each antibody interaction may not be required because any interaction that leads to a rigid and stable complex structure and/or provides an additional crystal contact could aid crystal formation. In addition, protein crystals grow at concentrations typically orders of magnitude higher than where Fabs are formed via disulphide bond formation between light and heavy chains. Open in a separate window Fig. 2 A schematic of the Fab phage library construct.
Specific buffers were used for each target membrane protein and are referred to as Tg1 cells (OD600= 0