Progress in the study of normal biological processes, or the mechanism of disease, has traditionally been hampered by the immense challenge in identifying the specific binding partners for a natural ligand or pathogen. Similarly, in drug discovery, the success of phenotypic screening approaches ultimately relies on deconvoluting the targets of any phenotypic molecules before these leads can be progressed.
In the case of antibodies or peptides (i.e., biologics), the binding targets will generally be receptors or other types of cell surface protein. Until recently, the hit rate for accurately identifying a cell surface protein interaction was approximately 10% using techniques such as standard protein chips or immunoprecipitation and mass spectrometry-based methodologies. However, a validated high-throughput approach—using a cell microarray technology developed by Retrogenix—has now increased the likelihood of success to more than 60% and has already contributed to breakthroughs in malaria research and the deconvolution of receptor(s) for many antibody ligands.
The elegance of the Retrogenix microarray system lies in expressing full-length human membrane proteins in the context of human cells. This allows for normal trafficking of the proteins to the cell surface, correct folding in the plasma membrane, and natural post-translational modification. Once expressed in their native state on the cell surface, these proteins are then available to interact with the test ligand. Representation of a large proportion of the plasma membrane proteome in a physiologically relevant system underpins the success of this technology in detecting specific hits for a variety of applications.
This tool utilizes a proprietary library of expression vectors containing open reading frames (ORFs) encoding more than 3,500 full-length human plasma membrane proteins, as well as a green fluorescent protein (GFP). Each vector is combined with a lipid and the complexes are spotted in distinct locations on glass slides. Human cells are then grown to confluence over the spotted slides.
Reverse-transfection of the cells sitting over each expression vector results in overexpression of each respective membrane protein, along with GFP. The ligand (e.g., protein, virus, phenotypic molecule) is applied to the cell microarrays and putative receptor targets identified by analyzing “gain-of-binding” using an appropriate detection system (Figure 1).
The GFP acts to determine spot coordinates, as well as provide a quality control to ensure that the minimum transfection threshold has been exceeded. Typically, further tests are then undertaken to determine that the receptor “hit” is reproducible and confirm specificity to the test ligand.