Fig. 3: Directed evolution of microID. | Communications Biology

Fig. 3: Directed evolution of microID.

From: Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Fig. 3

a Scheme of the biotinylation assay. MicroID random variants were surface-presented via Aga1p:Aga2p. The yeast surface was biotinylated in presence of ATP and biotin. The His-tag served as a presentation marker. Biotin was detected with a streptavidin-APC conjugate. b Density plots of surface presentation (Y-axis) vs. cell surface biotinylation (X-axis). The error-prone PCR based-yeast surface display library was screened by stepwise decreased biotinylation time. Upper row: Comparison of microID with the initial library (R1) after a 17 h biotinylation assay. Bottom row: same with the Round 3 (R3) library after a 10 min biotinylation assay. c Single clone analysis from the Round 3 library. d Positions of the mutated residues (structure based on PDB: 3EFR processed with the program ChimeraX). e Activity measurement of microID variants with an ELISA-based biotinylation assay. Measurements were performed in five individual experiments in triplicates or quadruplicates. Norm. activity refers to the microID absorbance at 405 nm set to 100%. Error bars indicate standard deviations.

Back to article page