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J Chromatogr A. 2014 Apr 11;1337:95-105. doi: 10.1016/j.chroma.2014.02.029. Epub 2014 Feb 19.

A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins.

Author information

1
Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany. Electronic address: sfrey@gwdg.de.
2
Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany. Electronic address: goerlich@mpibpc.mpg.de.

Abstract

Engineered protein tags that confer specific binding to standardized affinity resins have revolutionized recombinant protein purification. Ideally, these tags should, however, be removed during or following purification to restore an authentic N-terminus. We introduce here a new set of proteases and corresponding protease recognition modules that are optimally suited for this purpose: a SUMO-specific and a NEDD8-specific protease from Brachypodium distachyon (bdSENP1 and bdNEDP1), the NEDP1 protease from Salmo salar (ssNEDP1), Saccharomyces cerevisiae Atg4p (scAtg4) and Xenopus laevis Usp2 (xlUsp2). These new proteases are highly specific and cleave tags from a 50-fold (xlUsp2) to 10,000-fold (bdSENP1) molar excess of substrate per hour at 0°C. They are thus up to 1000-fold more active than TEV protease. The most efficient protease, bdSENP1, is even more active and far more salt tolerant than its yeast ortholog scUlp1, allowing efficient tag removal also in high salt buffers containing, e.g. 1M NaCl. ssNEDP1 is distinguished by an exceptional salt tolerance, and a considerable tolerance toward charged and bulky residues in the P1' position. xlUsp2 is unique in that it can restore, with low efficiency though, an N-terminal proline. As shown in the accompanying paper (S. Frey, D. Görlich, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.02.029), the orthogonality between bdSENP1, NEDP1, scAtg4 and xlUsp2 can be exploited for purifying multi-subunit protein complexes of defined stoichiometry.

KEYWORDS:

Affinity tag; NEDD8; P(1)' promiscuity; SUMO; Salt tolerance; Tag-removing protease

PMID:
24636565
DOI:
10.1016/j.chroma.2014.02.029
[Indexed for MEDLINE]
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