Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers

Sandy Schmidt, Mark Dörr, Uwe T. Bornscheuer

Publikation: Beitrag in Buch/Bericht/KonferenzbandBeitrag in Buch/BerichtForschungBegutachtung

Abstract

Escherichia coli (E. coli) as heterologous host enables the recombinant expression of the desired protein in high amounts. Nevertheless, the expression in such a host, especially by utilizing a strong induction system, can result in insoluble and/or inactive protein fractions (inclusion bodies). Furthermore, the expression of different enzyme variants often leads to a diverse growth behavior of the E. coli clones resulting in the identification of false-positives when screening a mutant library. Thus, we developed a protocol for an optimal and reproducible protein expression in microtiter plates showcased for the expression of the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. By emerging this protocol, several parameters concerning the expression medium, the cultivation temperatures, shaking conditions as well as time and induction periods for CHMO were investigated. We employed a microtiter plate shaker with humidity and temperature control (Cytomat™) (integrated in a robotic platform) to obtain an even growth and expression over the plates. Our optimized protocol provides a comprehensive overview of the key factors influencing a reproducible protein expression and this should serve as basis for the adaptation to other enzyme classes.

Originalspracheenglisch
TitelMethods in Molecular Biology
Herausgeber (Verlag)Humana Press Inc.
Seiten145-156
Seitenumfang12
Band1685
DOIs
PublikationsstatusVeröffentlicht - 1 Jan 2018

Publikationsreihe

NameMethods in Molecular Biology
Band1685
ISSN (Print)1064-3745

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Libraries
Escherichia coli
Enzymes
Growth
Proteins
Acinetobacter
Temperature
Inclusion Bodies
Robotics
Humidity
Clone Cells
cyclohexanone oxygenase

Schlagwörter

    ASJC Scopus subject areas

    • !!Molecular Biology
    • Genetik

    Dies zitieren

    Schmidt, S., Dörr, M., & Bornscheuer, U. T. (2018). Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers. in Methods in Molecular Biology (Band 1685, S. 145-156). (Methods in Molecular Biology; Band 1685). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7366-8_8

    Library growth and protein expression : Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers. / Schmidt, Sandy; Dörr, Mark; Bornscheuer, Uwe T.

    Methods in Molecular Biology. Band 1685 Humana Press Inc., 2018. S. 145-156 (Methods in Molecular Biology; Band 1685).

    Publikation: Beitrag in Buch/Bericht/KonferenzbandBeitrag in Buch/BerichtForschungBegutachtung

    Schmidt, S, Dörr, M & Bornscheuer, UT 2018, Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers. in Methods in Molecular Biology. Bd. 1685, Methods in Molecular Biology, Bd. 1685, Humana Press Inc., S. 145-156. https://doi.org/10.1007/978-1-4939-7366-8_8
    Schmidt S, Dörr M, Bornscheuer UT. Library growth and protein expression: Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers. in Methods in Molecular Biology. Band 1685. Humana Press Inc. 2018. S. 145-156. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7366-8_8
    Schmidt, Sandy ; Dörr, Mark ; Bornscheuer, Uwe T. / Library growth and protein expression : Optimal and reproducible microtiter plate expression of recombinant enzymes in E. coli using MTP Shakers. Methods in Molecular Biology. Band 1685 Humana Press Inc., 2018. S. 145-156 (Methods in Molecular Biology).
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