Overproduction in Escherichia coli and purification of Epstein-Barr virus EBNA-1.
Epstein-Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein-Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni-NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni-NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).
Duellman
Sarah J
SJ
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA. sjduellman@wisc.edu
Burgess
Richard R
RR
D002846:Chromatography, Affinity; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D054334:Viral Regulatory and Accessory Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D019309:Epstein-Barr Virus Nuclear Antigens; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; C417029:EBV-encoded nuclear antigen 1