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Library Growth And Protein Expression: Optimal And Reproducible Microtiter Plate Expression Of Recombinant Enzymes In E. Coli Using MTP Shakers.

S. Schmidt, M. Dörr, U. Bornscheuer
Published 2018 · Chemistry, Medicine

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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.
This paper references
10.1016/j.enzmictec.2013.03.011
Direct biocatalytic one-pot-transformation of cyclohexanol with molecular oxygen into ɛ-caprolactone.
Svenja Staudt (2013)
10.1016/S0141-0229(00)00320-3
Large scale production of cyclohexanone monooxygenase from Escherichia coli TOP10 pQR239.
S. D. Doig (2001)
10.1002/anie.201410633
An enzyme cascade synthesis of ε-caprolactone and its oligomers.
S. Schmidt (2015)
10.1126/SCIENCE.1093857
Environmental Genome Shotgun Sequencing of the Sargasso Sea
J. Venter (2004)
10.1016/J.PEP.2005.01.016
Protein production by auto-induction in high density shaking cultures.
F. W. Studier (2005)
10.1016/j.tibtech.2008.03.004
New opportunities for biocatalysis: making pharmaceutical processes greener.
J. Woodley (2008)
10.1007/S00449-005-0422-4
Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis
C. Ferreira-Torres (2005)
10.1002/BIT.10344
The use of microscale processing technologies for quantification of biocatalytic Baeyer-Villiger oxidation kinetics.
S. D. Doig (2002)
10.1023/A:1005610815727
Production of cyclohexanone monooxygenase from Acinetobacter calcoaceticus for large scale Baeyer–Villiger monooxygenase reactions
S. Barclay (2004)
10.1002/bit.25925
Fully automatized high‐throughput enzyme library screening using a robotic platform
M. Dörr (2016)
10.1002/ANGE.201409227
Introducing an in situ capping strategy in systems biocatalysis to access 6-aminohexanoic acid.
Johann H. Sattler (2014)
10.1021/bp030044m
Understanding and Improving NADPH‐Dependent Reactions by Nongrowing Escherichia coli Cells
Adam Z. Walton (2004)
10.1002/BIT.20869
On oxygen limitation in a whole cell biocatalytic Baeyer–Villiger oxidation process
C. Baldwin (2006)
10.1021/bp010177c
An Efficient Enzymatic Baeyer–Villiger Oxidation by Engineered Escherichiacoli Cells under Non‐Growing Conditions
Adam Z. Walton (2002)
10.1016/j.cbpa.2009.01.018
Biocatalysis in development of green pharmaceutical processes.
Junhua Tao (2009)
10.1021/bp0200954
Reactor Operation and Scale‐Up of Whole Cell Baeyer‐Villiger Catalyzed Lactone Synthesis
S. D. Doig (2002)
10.1007/s00253-007-1016-7
Enhanced production of ɛ-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene
Won-Heong Lee (2007)
10.1038/nature11117
Engineering the third wave of biocatalysis
U. Bornscheuer (2012)



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