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ACS Comb Sci. 2018 Apr 9;20(4):203-211. doi: 10.1021/acscombsci.7b00153. Epub 2018 Feb 2.

Sortase-Mediated High-Throughput Screening Platform for Directed Enzyme Evolution.

Author information

1
DWI-Leibniz-Institute for Interactive Materials , Forckenbeckstra├če 50 , 52074 Aachen , Germany.
2
Institute of Biotechnology , RWTH Aachen University , Worringerweg 3 , 52074 Aachen , Germany.

Abstract

Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (application 1) and to perform directed laccase evolution through a semipurification process in 96-well microtiter plate (MTP) (application 2). A semipurification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI, which acts as adhesion promoter. To validate the SortEvolve screening platform for both applications, three site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) from Staphylococcus aureus (application 1) and two SSM libraries of the copper efflux oxidase (CueO laccase) from Escherichia coli (application 2) were generated at literature reported positions. After screening and rescreening, an array of Sa-SrtA variants (including the previously reported P94S, D160N, and D165A) and CueO variants (including the previously reported D439A and P444A) were identified. Further recombinant Sa-SrtA variant P94T/D160L/D165Q and CueO variant D439V/P444V were characterized with 22-fold and 103-fold improvements in catalytic efficiency compared with corresponding wild-types, respectively. An important advantage of the SortEvolve screening platform in comparison to many MTP-based screening systems is that the background noise was minimized (decreased 20-fold; application 2) due to the employed semipurification process. In essence, SortEvolve provides a universal surface-functionalized screening platform for sortases and enzymes in which especially background activity can be minimized to enable successful directed evolution campaigns.

KEYWORDS:

CueO; LCI; SrtA; anchor peptide; directed evolution; laccase

PMID:
29363945
DOI:
10.1021/acscombsci.7b00153
[Indexed for MEDLINE]

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