Mutant TEV proteases for enhanced protein cleavage

This invention describes mutant TEV proteases with enhanced activity for cleaving specific amino acid sequences. These proteases are useful for removing purification tags from fusion proteins and antibodies in lab settings.

Background

Protein purification is a cornerstone of biological research and drug development. The ability to isolate and study specific proteins relies heavily on techniques that can separate the target protein from complex mixtures. Affinity tags, short amino acid sequences added to a protein of interest, have revolutionized protein purification by providing a handle for selective capture. However, the removal of these tags after purification often requires highly specific proteases that cleave at defined sites without degrading the target protein.

Current protease-based tag removal strategies suffer from several limitations. Wild-type proteases often exhibit broad substrate specificity, leading to unwanted cleavage of the target protein. While engineered proteases with improved specificity exist, they may suffer from low catalytic efficiency, requiring high enzyme concentrations or long incubation times that can be detrimental to protein stability and experimental workflows. Additionally, some proteases are prone to autolysis, degrading themselves over time and reducing their effectiveness. These limitations underscore the need for improved proteases with enhanced specificity, catalytic efficiency, and stability for robust and efficient protein purification.

Technology description

This technology involves mutant TEV proteases engineered for enhanced catalytic efficiency and activity in cleaving specific amino acid sequences, such as ENLYFQG or ENLYFQS. Derived from the Tobacco Etch Virus (TEV), these proteases incorporate specific amino acid substitutions achieved through directed evolution. These mutations result in a protease with a higher turnover rate and reduced Km, significantly boosting its efficiency compared to previous TEV protease variants. This makes them highly valuable for laboratory applications like cleaving fusion proteins and removing purification tags from fusion proteins or antibodies.

What sets this technology apart is the engineering process employed and the resulting protease’s performance. The YESS 2.0 system, a yeast-based platform, enabled the creation of a highly efficient TEV variant, eTEV. This variant exhibits an eight-fold increase in catalytic efficiency compared to its parent, TEV-S219P, and surpasses the performance of other engineered TEVs like uTEV3 in protein cleavage assays. Notably, eTEV achieves this through a combination of reduced Km and increased turnover rate (kcat), indicating enhanced substrate binding and cleavage. This combination of features makes eTEV a superior tool for protein research and related applications.

Benefits

Enhanced catalytic efficiency and activity compared to previous TEV protease variants
Higher turnover rate (kcat) and reduced Km, making it more efficient
Useful for cleaving fusion proteins and removing purification tags
Specifically designed for cleaving amino acid sequences like ENLYFQG or ENLYFQS
Can be produced in E. coli, potentially as fusion proteins with tags for purification