PROFICS: A bacterial selection system for directed evolution of proteases

Abstract

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

Caspase-Based Fusion Protein Technology: Substrate Cleavability Described by Computational Modeling and Simulation

“…Those sites were mutated through all 20 amino acids to calculate their free energies. The process was reiterated with two different substrates, with Ile and Pro as the P1′ amino acid, respectively, and the in-silico calculations were confirmed experimentally …”

“…The process was reiterated with two different substrates, with Ile and Pro as the P1′ amino acid, respectively, and the in-silico calculations were confirmed experimentally. 20 The ability of the CASPON enzyme to cleave the CASPONtag was tested using small peptides and the small protein ubiquitin-conjugating enzyme E2, in which the P1′ amino acid was replaced with all 20 proteogenic amino acids. The cleavability was found to function to a satisfying degree for all amino acids, with some working better than others.…”

“…The process was reiterated with two different substrates, with Ile and Pro as the P1′ amino acid, respectively, and the in-silico calculations were confirmed experimentally. 20 …”

Automated in vivo enzyme engineering accelerates biocatalyst optimization

“…Among bacterial species, E. coli continues to be a preferred option, and the principle of increased fitness over time in the presence of selective pressure has been exploited extensively—epitomized by the classical long-term evolution experiment (LTEE), where cells evolved to optimize carbon utilization pathways towards maximizing growth over 50,000 generations 121 . Building on this notion, and just to mention some key studies over the last 5 years, E. coli has been used for the selection and evolution of the activity of several enzymes (e.g., proteases 122 , deaminases 123 and formate dehydrogenases 80 ) and enzymes displaying emergent properties (either natural or engineered, e.g., using non-canonical redox cofactors 124 , 125 ). S. cerevisiae has been likewise used to evolve bacterial enzymes, e.g., an efficient tryptophan synthase from Thermotoga maritima using OrthoRep 103 .…”

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli

“…One such tag removal system is the recently developed CASPON technology, which is based on a modified human caspase-2 especially suited for the production of N-terminally tagged fusion proteins [ 30 ]. Its main advantages are the high activity, specificity, and good manufacturability of the enzyme, as well as the small size of the recognition sequence [ 30 , 31 , 32 , 33 ]. Great flexibility in the selection of suitable POIs is provided by the CASPON enzyme, due to the ability to cleave before any amino acid on the C-terminus of its recognition site [ 30 , 33 ].…”