Promega provides 10 96-well master plates containing plasmid-based cDNAs and 10 96-well Gold TnT SP6 Express 96 plates. Users simply transcribe and translate an aliquot from each master plate well using the TnT system. They then identify the presence of the protein activity using a detection assay, and subdivide the cDNAs in positive wells until they isolate the appropriate genes.
According to Michael Zwick, Promega's proteomics technology manager, the Proteolink system has two major benefits. The first is cost-effectiveness, Zwick says, and the second is flexibility. "Because it's in vitro, researchers can identify proteins that couldn't be identified using other systems, and they have the flexibility of altering conditions in ways that cell-based systems don't allow." Researchers could, for example, adjust components to identify optimal conditions for a protein's activity.
Zwick says that Promega used human adult brain for the cDNA library because the tissue expresses a diverse set of genes encoding many biochemical activities. The cDNA library, created in partnership with Rockville, Md.-based OriGene Technologies, was rigorously size-selected and configured to have 100 cDNAs per well, according to Zwick. "This system is good for someone who is studying any basic biochemical properties," he says. "It can also be used as a primary screen for protein: protein interactions."
According to Gary Kobs, Promega product manager, the user identifies expressed proteins with a functional screen, and then matches the protein to its cDNA of origin in the Proteolink system. Researchers have used this approach to identify protease and kinase substrates, protein:protein interactions, and protein:nucleic acid interactions. They have also used this method to discover novel genes with low sequence homology to other family members, but yet whose proteins have similar functions. Scientists can potentially adapt the system to screen for protein interactions in which the "bait" is a multimeric enzyme complex, ribosome, virus, or even a cellular organelle.