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. Author manuscript; available in PMC: 2021 Jul 11.
Published in final edited form as: Nat Chem Biol. 2021 Jan 11;17(3):317–325. doi: 10.1038/s41589-020-00706-1

Extended Data Fig. 4: HPLC assay of LMVGG activity.

Extended Data Fig. 4:

In this assay, sortase-mediated transpeptidation of a chromophore-linked substrate (Abz-LMVGGK-Dnp) liberates free GK-Dnp, the formation of which can be followed by HPLC1. The round 2 consensus sequence (R2, 4S.6 + R94Y, S118I, A122W, D124G, G134R, and V189F mutations) did not show detectable activity in this assay. The first variants with detectable activity on LMVGG in this assay emerged from round 4; the most active of these variants (R4) displayed modest activity on Abz-LMVGGK-Dnp, with 1 μM of enzyme converting 5.8% of 10 μM substrate to product in two hours. Notably, this variant contained the V182A, T196S, and R197S mutations. By round 8, activity in this assay roughly doubled, with 1 μM of clone 8.5-H3 converting 10.4% of 10 μM substrate to product in two hours.