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*

*Korrespondierende/r Autor/in für diese Arbeit

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

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

ASJC Scopus subject areas

  • !!Molecular Biology
  • Genetik

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