화학공학소재연구정보센터
Protein Expression and Purification, Vol.63, No.2, 128-133, 2009
Large-scale Epstein-Barr virus EBNA1 protein purification
The protein-DNA and protein-protein interactions of Epstein-Barr virus nuclear antigen 1 (EBNA1) are known to play an important role in the many functions of this viral protein. Large quantities of pure EBNA1 protein would be useful in biochemical assays to elucidate such interactions. In particular, the crystal structure of the full-length protein would be important to show possible regions of interaction and/or post-translational modification. Recently, we described a novel approach to overexpress and purify EBNA1 from Escherichia coli; however, it is not ideal for large-scale production of EBNA1. We were able to optimize this protocol by (1) adding a polyethyleneimine precipitation step prior to Ni-NTA chromatography to reduce complexity of the sample and remove nucleic acid, (2) optimizing the Ni-NTA gradient to further separate EBNA1 from impurities, and (3) concluding with a MonoS cation-exchange chromatography step to further purify and concentrate EBNA1. We were able to recover 10-mg quantities of pure EBNA1 protein. (C) 2008 Elsevier Inc. All rights reserved.