Random Addressed Protein Arrays
In the last decade, sequencing technologies have seen prodigious improvements in throughput and costs yet, protein activity and enzymatic assays –and hence our ability to query gene function– have not enjoyed concomitant advances. We are interested in applying the high-throughput sequencing technologies to generate an array of clusters containing DNA templates for in vitro transcription/translation (IVTT) reactions. Our goal is to identify each template in the array by sequencing and then generate a high-density protein array by capturing in vitro translated proteins proximal to the template cluster.
Towards this goal we have established methods for clonal amplification of DNA on solid surfaces (solid-phase amplification and emulsion amplification), in vitro transcription and translation methods using home-made cellular extracts, and are working on strategies for capturing proteins on surfaces.
Notably, an array platform containing millions of features will allow an increase in throughput three orders of magnitude above that of current protein arrays. Such increased throughput will enable experiments aimed at mapping protein interactions, generating enzyme activity profiles with activity-based probes and identifying novel enzyme activities to be performed at new scales. The ability to perform massively parallel protein assays should open novel research opportunities in proteomics, metagenomics and directed protein evolution. In addition, such development would also show how high-throughput sequencing platforms can be integrated into new technologies for functional assays, with end-points distinct from sequencing reads.
•Carlos Araya and Doug Fowler
Malaria Protein Arrays
Malaria is one of the world’s most devastating human diseases, affecting an estimated 500 million people and resulting in 2.5 million deaths globally each year. It is caused by four species of the protozoan parasite, Plasmodium, with the most deadly being Plasmodium falciparum. Progress in understanding malaria has been limited by the parasite’s complex life cycle and by difficulties expressing and purifying functional P. falciparum proteins in heterologous systems. With the completion of the P. falciparum genome sequence, a useful tool would be a protein array in which individual parasite proteins are displayed and available for functional assays.
To prepare a P. falciparum protein array, we are using 1000 P. falciparum open reading frames generated by the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. These ORFs will be translated in vitro and printed onto glass slides. Our goal is to use these protein arrays in assays that benefit from rapid, simultaneous and sensitive screening of large numbers of proteins. For example, antigen profiling assays can identify P. falciparum proteins that govern host immune responses against the pathogen. Another application for the array is the biochemical characterization of the “hypothetical” proteins that represent more than 60% of P. falciparum genome, many of which are unique to P. falciparum. To characterize these proteins we will profile arrayed proteins with fluorescent substrate profiling probes.
•Anastasia Gridasova
Identifying protein targets of ubiquitin ligases
Ubiquitin (Ub) is a 76 amino acid protein that when attached to a target protein can alter its fate in multiple ways. Ub is an essential signaling molecule in nearly every pathway in eukaryotic cells. The E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases act in concert in order to covalently attach a Ub moiety to the epsilon amine on a lysine side chain or less commonly, the free amino group at the N-terminus of a substrate protein via an isopeptide bond. The E3 gives the enzyme cascade substrate specificity. There are 35 E3s in S. cerevisiae and nearly 1000 putative E3s in the human genome. Many proteins important for human disease are E3s, such as the breast cancer-specific tumor suppressor BRCA1 (breast cancer-1) and the early onset-Parkinson’s disease protein Parkin. Therefore, knowing the specific protein substrates of individual E3 enzymes would be of great importance in medicine.
One approach I am taking to determine specific substrates of E3s is as follows:
Individual E3 ubiquitin ligases will be fused to the bacterial biotin ligase, BirA, which attaches biotin to proteins that contain a biotin-acceptor peptide. A second fusion protein will be generated where ubiquitin is fused to a biotin-acceptor peptide fragment. In this arrangement, when the biotin-acceptor/ubiquitin fusion protein is brought to the E3 ubiquitin ligase/BirA fusion protein, the attached BirA will biotinylate the ubiquitin, thus marking it as having been acted on by that specific ubiquitin ligase. Substrate proteins to which the biotinylated ubiquitin is subsequently attached can then be purified using streptavidin chromatography and identified by mass spectrometry. Applying this approach to each individual E3 ubiquitin ligase will provide a critically needed proteome-wide view of ubiquitin ligases and their substrates. Ultimately, I would adapt the system for the study of ubiquitin ligases in mammalian tissue culture cells.
•Lea Starita
