title: Efficient expression and purification of tag-free Epstein-Barr virus EBNA1 protein in Escherichia coli by auto-induction.
abstract: 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.
authors.affiliation: DNA Replication and Epigenetics Group, Research Unit Gene Vectors, Helmholtz Zentrum München, Germany.
authors.forename: Christoph-Erik
authors.initials: CE
authors.lastname: Mayer
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.forename: Aloys
authors.initials: A
authors.lastname: Schepers
mesh_terms: D002843:Chromatin / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D059748:Proteolysis
chemical_list: D002843:Chromatin; D019309:Epstein-Barr Virus Nuclear Antigens; D010450:Endopeptidases; C099488:TEV protease; C417029:EBV-encoded nuclear antigen 1; D005998:Glycine