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  Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases.

Frey, S., & Görlich, D. (2014). Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. Journal of Chromatography A, 1337, 106-115. doi:10.1016/j.chroma.2014.02.030.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0018-90DB-5 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0029-C76E-7
Genre: Journal Article

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1977176_Suppl.pdf (Supplementary material), 2MB
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 Creators:
Frey, S.1, Author              
Görlich, D.1, Author              
Affiliations:
1Department of Cellular Logistics, MPI for biophysical chemistry, Max Planck Society, ou_578574              

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Free keywords: Affinity tag, Controlled subunit stoichiometry, On-column cleavage, Protein complex, Tag-removing protease
 Abstract: Tag-free proteins or protein complexes represent certainly the most authentic starting points for functional or structural studies. They can be obtained by conventional multi-step chromatography from native or recombinant tag-free sources. Alternatively, they can be expressed and purified using a cleavable N-terminal affinity tag that is subsequently removed by a site-specific protease. Proteolytic tag-removal can also be performed "on-column". We show here that this not only represents a very efficient workflow, but also drastically improves the purity of the resulting protein preparations. Precondition for effective on-column-cleavage is, however, that the tag-cleaving protease does not bind the stationary phase. We introduce scAtg4 and xlUsp2 as very good and bdSENP1, bdNEDP1 as well as ssNEDP1 as ideal proteases for on-column cleavage at 4°C. Four of these proteases (bdSENP1, bdNEDP1, scAtg4, xlUsp2) as well as TEV protease display orthogonal, i.e. mutually exclusive cleavage specificities. We combined these features into a streamlined method for the production of highly pure protein complexes: Orthogonal affinity tags and protease recognitions modules are fused to individual subunits. Following co-expression or in-vitro complex assembly, consecutive cycles of affinity capture and proteolytic release then select sequentially for the presence of each orthogonally tagged subunit, yielding protein complexes of well-defined subunit stoichiometry.

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Language(s): eng - English
 Dates: 2014-02-192014-04-11
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.chroma.2014.02.030
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Title: Journal of Chromatography A
Source Genre: Journal
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Pages: - Volume / Issue: 1337 Sequence Number: - Start / End Page: 106 - 115 Identifier: -