A fluorescent protein exchange strategy for detecting protein-protein interactions and discovering proteinaceous inhibitors in Escherichia coli

• Author / Creator

  Enterina, Jhon Ralph De La Peña

• Green fluorescent protein (GFP), along with other GFP-like proteins, is one of the fundamental tools that are enabling the exciting current era of biological studies. The utility of GFP-like proteins as genetically encoded markers and sensors has provided us with powerful tools for tracking proteins and monitoring biochemical events important to health and diseases. While fluorescent proteins (FPs) are now considered indispensable in the field of bioimaging, protein engineers are striving to apply and/or evolve FPs for a more diverse range of novel and exciting applications. One of the products of these engineering efforts is fluorescent protein exchange (FPX) technology. This strategy relies on competitive interactions between two different colors of dimerization-dependent FPs (ddFPs). Specifically, a red “A” and a green “A” FP compete for binding to a dark “B” partner. In an effort to expand utility of FPX in detecting protein-protein interactions (PPIs), we describe the development of a new method that can be used to detect association and disruption of interacting proteins in bacterial colonies. This approach enables high-throughput screening in validating interacting partners and engineering better protein or peptide-based inhibitors for disrupting aberrant PPIs. In this thesis we describe the construction and characterization of several bacterial polycistronic vectors which provided us a simpler and more efficient system for co-expressing interacting proteins in Escherichia coli. This work yielded two bicistronic vectors and two tricistronic vector which are able to express two and three separate proteins simultaneously. Characterization revealed that all vectors maintained the inherent features of their parent pBAD vector, including the tightly-regulated expression. We also describe the construction of pFPX, a tricistronic vector derived plasmid that can co-express potentially interacting proteins as ddFP fusions. Using pFPX and the custom fluorescence imaging system, we have successfully monitored associations of E1/K1, p53 transactivation domain/HDM 2 and HRasWT/Raf-1WT RBD. We also established the applicability of our strategy in screening affinity-engineered interactions by library generation and screening. First, we randomly mutated the 89th amino acid of the nonbinding Raf-1R89L RBD mutant and screened for clones which rescued its binding of HRasWT. Second, we panned libraries of HRas extragenic suppressors and selected clones that gained affinity to Raf-1R89L RBD. And third, we designed and optimized 7-mer inhibitory peptides that selectively bind to the LPA2 interacting-PDZ domains of either NHERF-2 (PDZ 2) or MAGI-3 (PDZ 6).

• Subjects / Keywords

  • Fluorescent protein exchange
  • Protein-based inhibitors
  • Protein-protein interactions

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3736MF0M

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.