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%0 Thesis
%A Zou, Zhi
%T Directed sortase evolution for site-specific protein engineering and surface functionalization
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2019-02768
%P 1 Online-Ressource (146 Seiten) : Illustrationen
%D 2019
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2019
%X Sortase-mediated ligation (SML) has emerged as a improtant tool for site-specific bioconjugation in protein engineering and material functionalization. Sortase A from Staphylococcus aureus (Sa-SrtA) specifically recognizes an LPxTG (in which x means any amino acid) motif in the target protein 1 and cleaves the scissile amide bond between threonine and glycine. The generated thioester intermediate subsequently ligates to the target protein 2 with oligo glycine at N-terminal. Despite many highlights in applications, wild type Sa-SrtA suffers from several notable limitations (e.g. a relatively low catalytic efficiency (high Km (LPxTG) ≈ 6.5 mM), a strict specificity for the LPxTG motif and dependency on calcium cofactor). Directed evolution is a powerful tool to tailor enzyme properties towards user-defined goals. Directed sortase A evolution requires the development of a robust high-throughput assay which directly detects the formed conjugated products. Several conjugated product-based high-throughput screening strategies (e.g. cell surface display and in vitro compartmentalization) of Sa-SrtA have been established. Variants with enhanced activities, altered substrate specificities (rather than LPxTG motif) and calcium-independence were identified. However, these strategies are rather specific for engineering of one property of sortase A and usually limited in versatilities. For such purpose, it was essential to establish a general, robust and reliable screening system in microtiter plate (MTP) format which is applicable to perform directed Sa-SrtA evolution campaigns for different properties (e.g. thermos-stability and solvent resistance). In order to advance research of sortase engineering, this thesis was focused on the development of a general high-throughput screening system of sortase A, directed evolution of sortase A for efficient site-specific ligations in organic solvents, and applications of sortase A for covalent immobilization of multiple proteins on microgel. In the first section, a general directed sortase evolution platform (SortEvolve) was developed in in 96-well MTP made of polypropylene (PP-MTP). Two applications were carried out for SortEvolve. In Application 1, SortEvolve was validated for the directed Sa-SrtA evolution. In Application 2, SortEvolve was validated for the directed evolution of CueO laccase with minimized background noise (20-fold decreased). SortEvolve ensures a comparable amount/semi-purified enzyme through immobilization in PP-MTP. The latter is beneficial to avoid false positives during screening and also suited for directed evolution campaigns in which background activity (or noise) from crude lysate has to be minimized in order to identify beneficial variants In the next section, directed Sa-SrtA evolution campaign (KnowVolution) towards organic solvents was implemented. Organic solvents (e.g. DMSO, DMF) are routinely used to dissolve hydrophobic compounds. Engineering of Sa-SrtA for improved resistance/activity in organic co-solvents facilitates SML for more broad range of substrates. A random mutagenesis library (SeSaM library) of Sa-SrtA was screened in DMSO co-solvent by a modified SortEvolve protocol. Sa-SrtA variant M1 (R159G) with 2.2-fold improved resistance and variant M3 (D165Q/D186G/K196V) with 6.3-fold catalytic efficiency in 45
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2019-02768
%U https://publications.rwth-aachen.de/record/758220