Evaluating the Versatility of SpyTag002-SpyCatcher002 Reaction

Example 4

We tested the resilience of the reaction of SpyTag002 and SpyCatcher002 under a wide range of conditions. The above rate constants were calculated at pH 7, but reactivity was similar at pH 4 and slightly higher at pH 5 and 6 (FIG. 13A). Reaction was fast at 4, 25 and 37° C. (FIG. 13B). Reaction was relatively independent of buffer, with efficient reaction with phosphate, Tris or HEPES buffering, with relatively little dependence on specific monovalent or divalent anions or cations (FIG. 13C). Reaction of SpyTag002 and SpyCatcher002 tolerated well the presence of detergents Triton X-100 or Tween-20, giving a slight enhancement of reactivity (FIG. 13D). Reaction of SpyTag002 and SpyCatcher002 also tolerated over 3M urea (FIG. 13E).

SpyCatcher002 was selected on phage as an N-terminal fusion to pill. We confirmed that SpyCatcher002 also behaved well as a C-terminal fusion, showing efficient reaction of MBPx-SpyCatcher002 with SpyTag002-MBP (FIG. 14A). We validated that SpyTag002 reacted efficiently when fused at the N-terminus as SpyTag002-MBP (FIG. 12) or at the C-terminus as AffiEGFR-SpyTag002 (FIG. 14B).