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Mayer, C.E. ; Geerlof, A. ; Schepers, A.

Efficient expression and purification of tag-free Epstein-Barr virus EBNA1 protein in Escherichia coli by auto-induction.

Protein Expr. Purif. 86, 7-11 (2012)
DOI PMC
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
Epstein-Barr nuclear antigen 1 (EBNA1) is the essential Epstein-Barr virus (EBV) protein at the interface between the EBV genome and the host chromatin. It is EBNA1's task to guarantee replication and segregation of the multicopy closed circular viral genome in infected cells. While EBNA1's functions are relatively well understood, little is known about the molecular mechanisms of EBNA1 mediating chromatin tethering and DNA replication. To characterize those, purified EBNA1 would be a very useful tool in many different biochemical assays. For long, it was not possible to overexpress sufficient quantities of EBNA1 in Escherichia coli (E. coli) due to its rare codon usage, especially in the N-terminal part of the protein. Recently, some groups succeeded in purifying EBNA1 from bacteria using advanced inducible E. coli cells [1-3]. However, all purification procedures ended in a His-tagged version of EBNA1, which might influence EBNA1's function in biological assays. Therefore, we inserted a tobacco etch virus (TEV)-cleavage site between the N-terminal His-tag and the following open reading frame of EBNA1. Using sequential Ni-NTA and gel filtration columns and TEV protease-mediated cleavage upon autoinduction, we were able to purify functional EBNA1 protein featuring just a single additional, artificial N-terminal glycine residue. Following our simple and fast purification scheme we were able to synthesize 2mg of highly pure EBNA1 protein per liter culture.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter EBNA1; rare codon usage; his-affinity purification; EMSA; ORIGIN-BINDING-PROTEIN; DNA-BINDING; CRYSTAL-STRUCTURE; REPLICATION; OVERPRODUCTION; POLYMERASE; LINKING; DOMAIN; CELLS
Sprache englisch
Veröffentlichungsjahr 2012
HGF-Berichtsjahr 2012
ISSN (print) / ISBN 1046-5928
e-ISSN 1046-5928
Zeitschrift Protein Expression and Purification
Quellenangaben Band: 86, Heft: 1, Seiten: 7-11 Artikelnummer: , Supplement: ,
Verlag Elsevier
Begutachtungsstatus Peer reviewed
Institut(e) Research Unit Gene Vector (AGV)
Institute of Structural Biology (STB)
POF Topic(s) 30203 - Molecular Targets and Therapies
Forschungsfeld(er) Immune Response and Infection
Enabling and Novel Technologies
PSP-Element(e) G-501500-004
G-503000-001
G-503000-003
PubMed ID 22944205
DOI 10.1016/j.pep.2012.08.010
WOS ID WOS:000310654600002
Scopus ID 84866285009
Erfassungsdatum 2012-11-06
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