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[摘要]：Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-eided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LpIA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated. |
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