Ambergen Technology

Learn more about these technologies in our 2008 publication in Analytical Biochemistry "Photocleavage Based Affinity Purification and Printing of Cell-Free Expressed Proteins: Application to Proteome Microarrays".

Proteome Microarrays

Proteome microarrays facilitate a variety of important and highly multiplexed applications such as global mapping of protein-protein interactions in cellular pathways, proteome-wide detection of protein-drug interactions in drug discovery, determining kinase substrate preferences, evaluating antibody specificity and discovering new biomarkers, such as novel autoantigens (autoantibodies) useful in autoimmune and cancer diagnostics. However, the need to express, purify and print thousands of functional human proteins at high density on a microarray substrate presents challenges which limit their wide-spread availability and use. Rapid and facile cell-free protein expression has the potential to overcome many such problems, for instance, protein expression can be achieved in a few hours without the need for cellular transfection or cell culture, hence production is high throughout and highly automatable.

Ambergen's proprietary photocleavable linker (PC-Linker) and tRNA Mediated Protein Engineering (TRAMPE) technologies allow for rapid photo-purification and non-isotopic detection of cell-free expressed proteins, critical steps in the fabrication and readout of proteome microarrays. For instance, TRAMPE can be used to double-label cell-free expressed proteins with a fluorophore and photocleavable biotin (PC-Biotin), and proteins then readily photo-purified using the PC-Biotin, in a process termed PC-SNAG (Figure 1: "TRAMPE Based PC-SNAG"). Labeled and highly pure proteins can then be mechanically printed to planar surfaces to fabricate proteome microarrays (Figure 1; Center Panel). Figure 2A shows an actual protein microarray fabricated in this manner. In this case, a protein interaction assay between calmodulin and calcineurin is demonstrated.

As also depicted in Figure 1 ("Antibody Based PC-SNAG"), cell-free expressed proteins TRAMPE labeled with fluorescence only can be PC-SNAG purified using photocleavable antibodies (PC-Antibodies) and printed to microarrays. Figure 2B again shows an actual protein microarray fabricated in this manner, and its use in a protein interaction assay.

Proteome Bead-Displays

While the fabrication of cell-free expressed protein microarrays is highly automatable, production on a proteome-wide scale, by either cell-free or cellular expression, is still a daunting task that requires individual expressible plasmid clones, individual expression reactions and individual purifications for each member of the microarray.

Furthermore, once purified, proteins have to be serially printed using mechanical arrayers, which are relatively slow, problematic and can damage delicate proteins. All this contributes to the nearly prohibitive cost and low density of exiting proteome microarrays, which to date, still only cover relatively small fractions of the human proteome. Finally, these conventional microarrays, and the proteins for them, are usually mass-produced in bulk, leading to storage and dispensing issues which can result in protein damage and limit the ability to produce custom arrays of smaller protein subsets.

As a superior alternative, Ambergen is developing cell-free expressed proteome bead-display technologies, or Bead Sorted Libraries of in vitro Expressed Proteins (BS-LIVE-PRO) which are produced from Bead Sorted Libraries of in vitro Expressible DNA (BS-LIVE-DNA). BS-LIVE-DNA are produced in a single solid-phase PCR reaction and then converted to BS-LIVE-PRO on-demand, in a few hours, using multiplex cell-free protein expression (Figure 3). BS-LIVE-PRO can be decoded in a number of ways, including automated single-bead mass spectrometry, spectral bar coding or on a next-generation massively parallel DNA sequencer. Figure 4 shows proof-of-principal by demonstrating the fabrication of a model two protein BS-LIVE-PRO.

Photo-Printing of BS-LIVE-PRO

When beads carrying photocleavably tethered proteins are placed in close proximity to an activated planar surface, then illuminated, the proteins are photo-transferred directly to the surface to form discrete microarray spots whose dimensions match that of the beads; a process termed PC-PRINT (Figure 5). This novel printing technology, developed at Ambergen, has a number of advantages in the context of BS-LIVE-PRO. For instance, PC-PRINT of a BS-LIVE-PRO allows facile readout on a conventional microarray scanner and eliminates auto-fluorescence background from the beads themselves, as well as background caused by non-specific binding of contaminants, e.g. from the expression reaction, to the beads. This new approach provides an inexpensive method to fabricate very large scale, high density proteome "microarrays" from a BS-LIVE-PRO. As with BS-LIVE-PRO, several decoding methods are possible.

The dimensions of PC-PRINT spots are controlled by that of the bead, allowing spot densities orders of magnitude higher than conventional printing of protein arrays and microarrays (~100 micron spot minimum). As shown in Figure 6A, PC-PRINT spot sizes (e.g. 13 µm) and center-to-center spacing (e.g. 17 µm) can rival that of photolithographically produced DNA microarrays, and may have broad applications outside of proteomics. The sub-saturating spot density shown in Figure 6A corresponds to 13,000 spots/cm2 or ~240,000 spots on an entire 2.5 x 7.5 cm standard microarray substrate. Figure 6B demonstrates PC-PRINT in the context of cell-free protein expression, TRAMPE and protein interaction assays. PC-PRINT of DNA has also been achieved (not shown).