Flow cytometry based screening systems for directed evolution of proteases

  • Proteases are industrially important enzymes and advances in screening technologies such as flow cytometry based in vitro compartmentalization (IVC) offer novel opportunities to tailor protease properties by directed evolution. In this project two flow cytometry based screening systems (cell-free based ProCF-FCIVC and whole-cell based Pro-FCIVC) with a high throughput (>7500 s-1) have been developed for directed evolution of proteases. Firstly, system related technologies such as generation of w/o/w double emulsions, selection of a suitable substrate and calibration of the sorting performance of flow cytometry were investigated and optimized. Then a cell-free based ProCF-FCIVC was developed using the model protease M57, a serine protease isolated from a metagenome library. Different parameters like in vitro expression of protease, enzymatic reactions in emulsions, and recovery of sorted DNA variants were investigated. Sorting and subsequent activity analysis of the M57 reference library demonstrated a proof of concept of the developed ProCF-FCIVC system. For the whole-cell based Pro-FCIVC system, an extracellular protease deficient Bacillus strain together with a Subtilisin Carlsberg (SC) secretion system were chosen due to its low background activity of other non-target proteases. Several main challenges such as background of natural occurring proteases, high diversity library generation of Bacillus, sorting strategies and growth competition effects on sorting enrichment were studied and optimized. The developed whole-cell Pro-FCIVC system was then verified by screening an epPCR library with a high mutational load (~5 mutations per 1 kb) for inhibition resistance of SC towards the antipain dihydrochloride inhibitor. One variant (SCm2) with a 1.6-fold relative to wild-type SC improved resistance was isolated from a small population of active variants (315) validating the developed protease flow cytometry screening technology for increased inhibitor resistance.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Ran Tu
Referee:Ulrich Schwaneberg, Danilo Roccatano, Anett Kirschner
Advisor:Ulrich Schwaneberg
Persistent Identifier (URN):urn:nbn:de:101:1-2013052411239
Document Type:PhD Thesis
Language:English
Date of Successful Oral Defense:2010/07/26
Date of First Publication:2010/08/11
PhD Degree:Biochemical Engineering
School:SES School of Engineering and Science
Library of Congress Classification:T Technology / TP Chemical technology / TP248.13-248.65 Biotechnology / TP248.3 Biochemical engineering. Bioprocess engineering
Call No:Thesis 2010/13

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