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Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917
Zitatschlüssel ISI:000432417900002
Autor Fiege, Kerstin and Querebillo, Christine Joy and Hildebrandt, Peter and Frankenberg-Dinkel, Nicole
Seiten 2747-2755
Jahr 2018
ISSN 0006-2960
DOI 10.1021/acs.biochem.8b00242
Journal BIOCHEMISTRY
Jahrgang 57
Nummer 19
Monat MAY 15
Zusammenfassung Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.
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