Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Abstract

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.

A protease substrate profiling method that links site‐specific proteolysis with antibiotic resistance

“…2D). The relative ranking of Cml-sensitivity correlated well to previously reported processing efficiencies of the three substrates [18,19]. Moreover, a previously described [17] inactive mutant (D81N) of TEVp together with the reporter containing a TEVp-specific substrate peptide (CAT-subG-ssrA) did not support cell growth (Fig.…”

B cell recognition of myelin oligodendrocyte glycoprotein autoantigen depends on immunization with protein rather than short peptide, while B cell invasion of the CNS in autoimmunity does not

“…1A). An alternate form of the consensus cleavage site (ENLYFQ/G) (Kostallas et al, 2011) results in cleavage between the final glutamine and glycine, glycine being the first amino acid of MOG 1-125 . Therefore, TEV protease cleavage results in the removal of additional tag sequences, leaving pure MOG 1-125 extracellular domain without any residual amino acids.…”

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries

“…TEV-P recognizes the peptide sequence ENLYFQ↓S/G (27), where Q and S/G correspond to the P1 and P1′ residues, respectively. Even though cleavage sequence mapping studies using peptide combinatorial libraries have shown that the highest stringency was seen for Gln at P1 and that the P2, P4, and P5 positions can be occupied by a number of amino acids, no adventitious cleavage of full-length proteins at sites other than the canonical ENLYFQ↓S motif have been reported (28)(29)(30). We used the YESS system for the directed evolution of a TEV-P mutant library against a library of substrate constructs containing ENLYFX↓S, where X is any amino acid.…”