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

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

LanguageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages145-156
Number of pages12
Volume1685
DOIs
StatusPublished - 1 Jan 2018

Publication series

NameMethods in Molecular Biology
Volume1685
ISSN (Print)1064-3745

Fingerprint

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

Keywords

  • Baeyer-villiger monooxygenase
  • CHMO
  • Escherichia coli
  • High-throughput
  • Library growth
  • Microtiter plates
  • Reproducible protein expression
  • Screening

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 (Vol. 1685, pp. 145-156). (Methods in Molecular Biology; Vol. 1685). Humana Press Inc.. DOI: 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. Vol. 1685 Humana Press Inc., 2018. p. 145-156 (Methods in Molecular Biology; Vol. 1685).

Research output: Chapter in Book/Report/Conference proceedingChapter

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. vol. 1685, Methods in Molecular Biology, vol. 1685, Humana Press Inc., pp. 145-156. DOI: 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. Vol. 1685. Humana Press Inc.2018. p. 145-156. (Methods in Molecular Biology). Available from, DOI: 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. Vol. 1685 Humana Press Inc., 2018. pp. 145-156 (Methods in Molecular Biology).
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