Nucleic Acids Programmable Protein Array Core (NAPPA)
(Funding for this core ended March 1, 2011)
Former Core leader: Grant McFadden
University of Florida
The goal of the Core was to assist the deciphering of the molecular mechanisms of host responses to select pathogens using proteomics to uncover novel protein-protein interactions (PPIs). This project was originally pursued in collaboration with the Harvard Proteomics Facility, which was developing the NAPPA technology to create human and select bacterial pathogen protein microarrays. However, our pilot screenings yielded far too many false-positive PPI hits, using the NAPPA technology as adapted for multi-well format, and we switched to an alternative technology, called Alphascreen, that utilizes the same plasmid library that was created for NAPPA. In contrast to NAPPA, which utilizes in vitro expression of proteins, the Alphascreen method utilizes proteins expressed in vivo within mammalian cells. Our collaborators included several SERCEB investigators, including Drs. Mark Dennison and Ralph Baric (human coronaviruses), Dr. Margo Brinton (West Nile Virus [WNV]), Dr. Aravinda deSilva (Dengue viruses), Dr. Mark Heise (Chikungunya [CHIKV], Sindbis, Ross River, and Venezuelan Equine Encephalitis [VEE] viruses), and Dr. David Weiss (F. tularensis). We screened these pathogen proteins for novel PPIs against host protein targets that are elements of the human inflammasome complexes and the RIG-I pathway. Specifically, we:
- Created viral open reading frame (ORF) libraries from the MPXV genome (strain Zaire), Dengue (several serotypes), human SARS, West Nile, CHIKV, Sindbis, Ross River, and VEE viruses, as well as F. tularensis in Gateway plasmid expression vectors.
- Validated the expression of these encoded GST-tagged fusion proteins in reticulocyte lysates in vitro (for NAPPA) and in transfected mammalian cells in vivo (for Alphascreening).
- Initiated Alphascreen-based PPI screening of the expressed pathogen GST-tagged proteins with representative HA-tagged host protein members from the human inflammasome response pathway and selected RIG-I pathway family members.
- Started validating the expression of representative HA-tagged host protein members from the human IFN and TNF innate immune response pathways.