Membrane proteins are notoriously difficult to crystallize. It is often challenging to obtain structurally homogeneous proteins due to their conformationally heterogeneous nature. They need to be incorporated into artificial membranes that mimic their native state [1
], and the detergents used to solubilize the membrane proteins often lead to inactivity and aggregation [2
]. Moreover, membrane proteins have less surface area for crystal contacts due to micelle formation around the transmembrane segments. To make membrane proteins more amenable to crystallographic studies, these properties must be improved. Stable binding proteins can be used to improve some of these properties and have been shown to assist crystallization of membrane proteins [3
Binding proteins can be either antibodies/antibody fragments, or non antibody proteins. Non antibody binding proteins include avimers, which are artificial multidomain proteins derived from human extracellular receptor domains [9
], and DARPINs, which are derived from the ankyrin repeat motif present in numerous naturally occurring proteins [3
]. These non antibody binding proteins were engineered to create antibody like molecules with improved stability and production yield [3
]. Nevertheless, the most commonly used binding proteins in co-crystallization experiments are antibody fragments. They have a tendency to bind flexible regions of macromolecules [10
], which can result in a more rigid and structurally homogeneous population of the complex. Antibody fragments can also aid in obtaining a conformationally homogeneous population by recognizing a single conformation and locking the protein in that conformation [3
]. Furthermore, antibody fragments can provide additional crystal contacts of complexes. Such contacts are well depicted in the structures of the antibody fragment-membrane protein complexes such as the KscA potassium channel [5
] as well as soluble protein complexes [3
Antibody fragments exist in various forms, including nanobodies that are single variable domains, Fvs that are composed of variable domains of light and heavy chains, and Fragments antigen binding (Fabs) that are composed of variable domains and single constant domains of light and heavy chains. Despite that all three forms have been co-crystallized with proteins, Fabs are the most commonly used form. This may be explained by their physical properties. Fabs are more likely to provide sufficient additional crystal contacts especially compared to nanobodies (15kDa), which are a quarter the size of Fabs (55kDa). They are typically more stable than Fv fragments[13
]. Moreover, single chain Fvs, the most common form of Fv, can exist in a monomer-dimer equilibrium, which is not ideal for crystallization [14
]. Using standard procedures for generating a phage Fab library [15
], a fully human naïve B cell Fab library was produced as a rich source of potential binding partners to proteins of interest [16
Phage display is a biopanning technique, which uses affinity selection to generate recombinant antibodies against a variety of antigens [17
]. In M13 filamentous phage display, which is the most commonly used form, the DNA encoding antibody fragment library is fused into the minor coat protein gene, g3 in a cloning vector called a phagemid. The fusion protein is expressed and incorporated into phage particles assembled in Escherichia coli
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 E. coli
with the eluted phage and propagating the eluted phage (). Thus, the phage particles that display antigen-binding antibody fragments become enriched. Relevant phagemids can be extracted from the E. coli
cells infected with the eluted phage.
A panning scheme for membrane proteins
In comparison to 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. In vitro
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 [17
]. 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 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 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 [2
]. 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
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 Kd values. Therefore, subjecting all selected Fabs for analytical biophysical assays, though preferred, is not necessary. Grouping selected antibodies based on their relative affinities should be sufficient to expedite the identification of higher affinity binders. Rapid and reliable characterization of selected antibodies is an essential step for phage display becoming a more generally applicable procedure.
We have successfully identified Fabs for multiple membrane proteins, including transporters and a cation channel, by phage display panning of a human naïve B cell Fab phage library [16
]. Here we describe our general panning procedure for membrane proteins and our procedure to rapidly group the selected Fabs based on their relative affinities, using enzyme linked immunosorbent assay (ELISA) and small quantities of the unpurified Fabs. This procedure greatly speeds the prioritization of candidate binders to membrane proteins and will aid in structure determinations. In addition, these Fabs can prove useful for functional characterization since they can serve as agonists or antagonists of the target protein.