A New Highly Specific and Soluble Protease for Precise Removal of N‑Terminal Purification Tags

Biotherapeutics production has been significantly enhanced by affinity purification. After purification, however, it is often necessary to remove the affinity purification tag. Thus, we aim for a protease suitable for such a task with properties that include high production yields, good solubility and stability, high cleavage specificity, sufficiently fast turnover, and tolerance of the amino acid identity at the P₁' position (the C-terminus of the recognition site). Here, we describe the development and characterization of a novel protease named Con1, which is expressed and purified with high solubility and stability. The active site of Con1 harbors a Cys-His-Asp catalytic triad like most of the natural cysteine proteases from viral origins. This validates the optimum enzyme activity under ambient conditions, including physiological pH. Like the Turnip mosaic virus (TuMV) protease, Con1 recognizes the amino acid sequence EAVYHQ (P₆-P₁) and tolerates many different residues at the P₁' position. The studied 12 amino acids at the P₁' position represent the different grouping of hydrophobic (A, F, G, I, M, and W), polar uncharged (Q, S, and T), positively charged (R), and negatively charged (D and E) amino acids. Con1 shows faster kinetics than TuMV protease against selected P₁' substrates. As is typical for this class of viral proteases, Con1 does not cleave substrates with proline at the P₁' position. We also showed that Con1 efficiently removed the purification tags from representative pharmaceutical/research products such as StefinA, DARPin, and INF2α. Because the active site is close to the C-terminus, we found that Con1 is C-terminal sensitive. The activity is decreased upon trimming the last 7 residues; on the other hand, by cutting 13 residues, the catalytic efficiency has improved with a 2× lower K _m value than that of Con1. Overall, Con1 and its variant have suitable characteristics for biotech applications that will aid the biopharma industry.