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Directed evolution of the substrate specificity of biotin ligase
Author(s) -
Lu WeiCheng,
Levy Matthew,
Kincaid Rodney,
Ellington Andrew D.
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25176
Subject(s) - biotin , dna ligase , streptavidin , biochemistry , biotinylation , peptide , biology , substrate (aquarium) , escherichia coli , systematic evolution of ligands by exponential enrichment , enzyme , dna , microbiology and biotechnology , chemistry , gene , ecology , rna
We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA 6–40 and BirA 6–47 , showed 17‐fold and 10‐fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA 6–40 and the single substitution variant BirA M157T revealed that they had twoto threefold higher k cat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications. Biotechnol. Bioeng. 2014;111: 1071–1081. © 2013 Wiley Periodicals, Inc.