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The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

Frey S, Görlich D - PLoS ONE (2015)

Bottom Line: Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification.We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae.In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Logistik, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.

ABSTRACT
During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

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In-vitro cross-reactivity with other tag-cleaving proteases.A, Schematic representation of substrates used for (B) and (C). The TEV protease substrate contains an N-terminal His10-ZZ tag preceding the TEV protease recognition site. All other substrates follow the scheme described in Fig 2A, the protease recognition site, however, is replaced by the respective ubiquitin-like protein (UBL). B, Cross-reactivity between recombinant tag-cleaving proteases. bd, Brachypodium distachyon; tr, Triticum aestivum (summer wheat); xUb, Xenopus ubiquitin. Bands marked with an asterisk (*) originate from the respective protease. For complete gels see S6 Fig. C, Detailed titration analysis of cross-reactivity between Xenopus laevis (x), S. cerevisiae (sc) and wheat (tr) Atg4 homologs.
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pone.0125099.g008: In-vitro cross-reactivity with other tag-cleaving proteases.A, Schematic representation of substrates used for (B) and (C). The TEV protease substrate contains an N-terminal His10-ZZ tag preceding the TEV protease recognition site. All other substrates follow the scheme described in Fig 2A, the protease recognition site, however, is replaced by the respective ubiquitin-like protein (UBL). B, Cross-reactivity between recombinant tag-cleaving proteases. bd, Brachypodium distachyon; tr, Triticum aestivum (summer wheat); xUb, Xenopus ubiquitin. Bands marked with an asterisk (*) originate from the respective protease. For complete gels see S6 Fig. C, Detailed titration analysis of cross-reactivity between Xenopus laevis (x), S. cerevisiae (sc) and wheat (tr) Atg4 homologs.

Mentions: A crucial parameter for the practical application of tag-cleaving proteases is their substrate specificity. This parameter is especially important when mutually exclusive specificity ("orthogonality") to other proteases is strictly required, e.g. for purification of protein complexes with controlled subunit stoichiometry [34]. Also, it is important to know which host proteases could potentially cleave a given protease recognition site during expression. For practical applications, we were especially interested in the cross-reactivity of xAtg4B with the well-established TEV protease [52,53], scUlp1 [32], SUMOstar protease [35,36] and the recently described proteases bdSENP1, bdNEDP1, and xUsp2 [33,34]. In addition, we also included the wheat (Triticum) Atg4 ortholog (trAtg4). In order to analyze the specificity profiles of these proteases, we incubated a high concentration (20 μM) of each protease with 100 μM of each substrate protein (see Fig 8A) in all possible combinations for 3 h at 25°C (Fig 8B). For all proteases except TEV protease, these conditions correspond to a significant (>200- to 30 000-fold) over-digestion. Under these conditions, both xAtg4B14-384 and trAtg4 only cleaved substrates containing Atg8-like UBLs (xLC3B, xGATE16 or trAtg8), but none of the substrates dedicated to other proteases. Vice versa, substrates containing Atg8-like UBLs were exclusively cleaved by Atg4 proteases. Atg4 proteases and Atg8-type substrate proteins are therefore truly orthogonal to all other protease/substrate pairs analyzed. Within the Atg8-type substrates, interesting differences became apparent: While xLC3B was nearly exclusively recognized by xAtg4B14-384, both xGATE16 and trAtg8-containing substrates were in addition also cleaved by trAtg4.


The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.

Frey S, Görlich D - PLoS ONE (2015)

In-vitro cross-reactivity with other tag-cleaving proteases.A, Schematic representation of substrates used for (B) and (C). The TEV protease substrate contains an N-terminal His10-ZZ tag preceding the TEV protease recognition site. All other substrates follow the scheme described in Fig 2A, the protease recognition site, however, is replaced by the respective ubiquitin-like protein (UBL). B, Cross-reactivity between recombinant tag-cleaving proteases. bd, Brachypodium distachyon; tr, Triticum aestivum (summer wheat); xUb, Xenopus ubiquitin. Bands marked with an asterisk (*) originate from the respective protease. For complete gels see S6 Fig. C, Detailed titration analysis of cross-reactivity between Xenopus laevis (x), S. cerevisiae (sc) and wheat (tr) Atg4 homologs.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414272&req=5

pone.0125099.g008: In-vitro cross-reactivity with other tag-cleaving proteases.A, Schematic representation of substrates used for (B) and (C). The TEV protease substrate contains an N-terminal His10-ZZ tag preceding the TEV protease recognition site. All other substrates follow the scheme described in Fig 2A, the protease recognition site, however, is replaced by the respective ubiquitin-like protein (UBL). B, Cross-reactivity between recombinant tag-cleaving proteases. bd, Brachypodium distachyon; tr, Triticum aestivum (summer wheat); xUb, Xenopus ubiquitin. Bands marked with an asterisk (*) originate from the respective protease. For complete gels see S6 Fig. C, Detailed titration analysis of cross-reactivity between Xenopus laevis (x), S. cerevisiae (sc) and wheat (tr) Atg4 homologs.
Mentions: A crucial parameter for the practical application of tag-cleaving proteases is their substrate specificity. This parameter is especially important when mutually exclusive specificity ("orthogonality") to other proteases is strictly required, e.g. for purification of protein complexes with controlled subunit stoichiometry [34]. Also, it is important to know which host proteases could potentially cleave a given protease recognition site during expression. For practical applications, we were especially interested in the cross-reactivity of xAtg4B with the well-established TEV protease [52,53], scUlp1 [32], SUMOstar protease [35,36] and the recently described proteases bdSENP1, bdNEDP1, and xUsp2 [33,34]. In addition, we also included the wheat (Triticum) Atg4 ortholog (trAtg4). In order to analyze the specificity profiles of these proteases, we incubated a high concentration (20 μM) of each protease with 100 μM of each substrate protein (see Fig 8A) in all possible combinations for 3 h at 25°C (Fig 8B). For all proteases except TEV protease, these conditions correspond to a significant (>200- to 30 000-fold) over-digestion. Under these conditions, both xAtg4B14-384 and trAtg4 only cleaved substrates containing Atg8-like UBLs (xLC3B, xGATE16 or trAtg8), but none of the substrates dedicated to other proteases. Vice versa, substrates containing Atg8-like UBLs were exclusively cleaved by Atg4 proteases. Atg4 proteases and Atg8-type substrate proteins are therefore truly orthogonal to all other protease/substrate pairs analyzed. Within the Atg8-type substrates, interesting differences became apparent: While xLC3B was nearly exclusively recognized by xAtg4B14-384, both xGATE16 and trAtg8-containing substrates were in addition also cleaved by trAtg4.

Bottom Line: Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification.We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae.In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Logistik, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.

ABSTRACT
During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.

Show MeSH
Related in: MedlinePlus