Poster V4 - Characterization of Protein-Protein Interactions using Ribosome Display Protein Array

Introduction: Protein display technologies are instrumental in engineering protein-protein interactions for new therapeutic development. However, toxic or aggregation-prone proteins remain difficult to express and study with existing protein display methods. Ribosome display is a cell-free display method that has been successfully used to express challenging proteins, such as leucine-rich repeat containing receptors important in the central nervous system and in innate immunity [1]. In this work, we develop an extension of this method that enables direct tethering of ribosomal complexes displaying target proteins, overcoming the challenge of recombinant expression and purification of target proteins often faced in traditional protein display technologies.

Materials and

Methods: In ribosome display, mRNA templates are translated into protein using a PURExpress protein synthesis kit, a cell-free system containing purified E. coli components for transcription and translation. The templates contain a C-terminal spacer sequence lacking a stop codon based on the TolA protein that extends the target protein out of the ribosomal tunnel to allow for proper folding without releasing it from the ribosome. To synthesize target protein for immobilization in the protein array, mRNA templates were first generated using polymerase chain reaction (PCR) amplification and in vitro transcription. We then investigated three different strategies for tethering the complex. For the first immobilization strategy, hairpins were introduced in the PCR step that were specific to an RNA binding protein, MS2. For the second strategy, the 5’ end of the mRNA template was directly biotinylated using enzymatic reactions and a biotin-maleimide label. For the third strategy (Fig. 1A), biotinylated DNA oligonucleotides were annealed to a complementary sequence on the 5’ end of the mRNA template, and the excess oligonucleotides were removed by column purification. The mRNA templates were translated into protein to generate target-displaying ribosomal complexes. These complexes are then immobilized to a MS2 coated well plate (strategy 1) or a neutravidin coated well plate via the biotin-neutravidin interaction (strategies 2-3). Functionality of the target complexes was verified with RT-qPCR and ELISA.

Results, Conclusions, and Discussions: While the first two immobilization strategies inefficiently or non-specifically immobilized ribosomal complexes, the third strategy of annealing labeled oligonucleotides to the mRNA template prior to translation enabled fast and specific immobilization of complexes to neutravidin coated surfaces. The presence of the mRNA in the immobilized target was verified with RT-qPCR (Fig. 1B). The displayed protein containing a FLAG-epitope tag was shown to functionally interact with anti-FLAG antibodies by detecting antibody binding with ELISA (Fig. 1C). Overall, we demonstrated a method to tether target proteins to a well surface for studying protein-protein interactions that does not require recombinant expression and purification of the target protein. This method of directly tethering ribosomal complexes could be used in future studies to express difficult targets involved in pathogenesis and design new protein therapeutics against them.

Acknowledgements (Optional): This work was supported by a Graduate Research Fellowship from NSF and grant R35GM136309 from NIH.

References: [1] Schimmele et al., J. Mol Biol. 2005, v. 352, 229-241