Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Cell-free based approach for rapid detection of protein-protein interactions (#282)

Shayli Varasteh Moradi 1 , Wayne A. Johnston 1 , Dejan Gagoski 1 , Kirill Alexandrov 1
  1. Institute for molecular biosciences, The University of Queensland, St. Lucia, QUEENSLAND, Australia

Protein–protein interactions (PPIs) play a pivotal role in most biological processes and the majority of proteins perform their function as parts of a protein interaction network or a stable protein complex. Therefore, the ability to rapidly analyse PPIs is critical for elucidation of their function. In vitro protein production holds the promise of accelerating protein-protein interaction analysis. We present a rapid in vitro protein-protein interaction analysis method based on AlphaLISA technology combined with Leishmania tarentolae cell-free protein production (LTE) system. Cell-free protein expression allows the rapid production of recombinant proteins in a multiplexed format. Among the available in vitro expression systems, LTE offers several advantages over other eukaryotic cell-free systems. It is based on a fast growing fermentable organism that is inexpensive in cultivation and lysate production. High integrity of proteins produced in this system and the ability to co-express multiple proteins makes it a desirable method for screening protein interactions. Following the translation of protein pairs in LTE system, the physical interaction between proteins can be analysed by AlphaLISA. This assay is performed using unpurified in vtiro translation reaction and therefore can be readily multiplexed. This method can be used in various research applications such as epitope mapping, antigen-antibody analysis and protein interaction network mapping. We applied the developed pipeline to analyse the viral protein interaction network of Zika virus. The viral proteins were co-expressed pair-wise in LTE and all possible interactions among viral proteins were tested using AlphaLISA. The assay resulted in identification of 54 intra-viral protein-protein interactions from which 19 binary interactions were found to be novel. The presented technique provides a powerful tool for rapid analysis of protein-protein interaction with high sensitivity and throughput.