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Institute of Biological Chemistry, National Core Facilities of High Throughput Protein Production, Academia Sinica, Taipei 115, Taiwan
(RECEIVED September 16, 2004; FINAL REVISION December 9, 2004; ACCEPTED December 9, 2004)
Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self-cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.
Keywords: tobacco etch virus protease; sticky-end PCR; fusion protein approach
Abbreviations: TEVP, tobacco etch virus protease • rsTEV, TEV recognition site • Sso, Sulfolobus solfataricus • MBP, maltose binding protein • Trx, thioredoxin • GST, glutathione S-transferase • CBP, calmodulin binding protein; His6, hexahistidine tag • FC, fusion carrier
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041129605.
Reprint requests to: Ting-Fang Wang or Andrew H.-J. Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; e-mail: tfwang{at}gate.sinica.edu.tw or ahjwang{at}gate.sinica.edu.tw; fax: +886-2-27889759.
1 These authors contributed equally to this work.
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