Functional protein arrays consist of thousands of proteins immobilized on a glass slide to facilitate the study of proteome-scale protein-interaction networks, autoantibody detection for clinical diagnosis, detection for infectious diseases, and vaccine research. Despite great promise, protein arrays have not been widely accepted because of technological hurdles including generating protein content, challenges in protein immobilization, and the need for complex robotics.
To overcome these problems, Promega offers a simple way to create medium-throughput custom protein arrays using the HaloLink™ Protein Array System. HaloLink is based on HaloTag®, a 33 kDa protein tag that forms a covalent bond with its ligand. HaloTag is multifunctional and has been used for protein expression and purification, cell-imaging, protein-immobilization, and protein-interaction analyses.
HaloLink Protein Array Systems combine protein-expression systems, a novel glass slide coating, and HaloTag technology to simplify the process of making custom protein arrays. This tutorial will focus on cell-free protein-expression systems for generating protein content, but HaloLink arrays have been successfully validated using cell-based expression systems like mammalian, E.coli, and insect cells.
Cell-free protein-expression systems based on coupled transcription-translation express proteins directly from DNA templates, are easy to use, rapid, and ideal for high-throughput formats. In addition, they can be used to express toxic, membrane, and insoluble proteins and allow insertion of non-natural amino acids containing fluorescent or other reactive labels.
The HaloLink Protein Array System uses polyethylene glycol (PEG)-coated glass slides for protein capture. PEG-based hydrogels resist nonspecific adsorption of unwanted protein and minimize surface-induced denaturation of immobilized proteins. PEG-coated glass slides activated with HaloTag ligands (HaloLink slides) are used to capture proteins-of-interest (POI) fused to HaloTag that are expressed in cell-free protein-expression systems.
Attachment through HaloTag is covalent, and POI are oriented for maximum functional activity, but more importantly, no prior protein purification is necessary. Although, other protein tags like polyhisitidine and GST can be used for orienting proteins at the surface, leaching due to the reversible nature of binding limits their utility. Finally, a silicone gasket that can be attached to the slide creates 50 wells on the glass slides allowing multiple assays to be performed manually on the same slide without any specialized equipment.
The combination of cell-free expression systems and HaloTag attachment chemistry allows users to create a medium-throughput custom array and perform downstream protein-interaction applications in less than eight hours (Figure 1A).