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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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Stability of UBL fusions in eukaryotic lysates and in S. cerevisiae.A, Schematic representation of substrates used for (B). B, Stability of protease substrates in cell extracts. Note that in wheat germ extract no proteolytic fragments originating from SUMOstar-, xLC3B- or xGATE16-containing substrates can be detected. For complete blots and stained membranes see S10 Fig. C, Schematic representation of substrates used for expression in S. cerevisiae (D) harboring an N-terminal ZZ-tag, an ubiquitin-like protein (UBL) and a C-terminal Citrine. D, In-vivo stability of protease substrates in S. cerevisiae. Indicated protease substrates were over-expressed in a S. cerevisiae strain constitutively expressing H2B-CFP. Total cell lysates were analyzed by Western blot with antibodies recognizing the ZZ-tag (upper panel) or Citrine and CFP (middle panel). Equal loading was confirmed by staining the membrane after blotting (lower panel). Bands marked with an asterisk (*) originate from ZZ-tagged proteins cross-reacting with the anti-Citrine/CFP antibody. For complete original blots and stained membranes see S11 Fig. E, Cleavage of UBL substrates in extracts and in S. cerevisiae. ++, highly efficient cleavage; +, cleavage;–, traces cleaved;––, no cleavage; n.d.: not determined; 1, data not shown.
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pone.0125099.g009: Stability of UBL fusions in eukaryotic lysates and in S. cerevisiae.A, Schematic representation of substrates used for (B). B, Stability of protease substrates in cell extracts. Note that in wheat germ extract no proteolytic fragments originating from SUMOstar-, xLC3B- or xGATE16-containing substrates can be detected. For complete blots and stained membranes see S10 Fig. C, Schematic representation of substrates used for expression in S. cerevisiae (D) harboring an N-terminal ZZ-tag, an ubiquitin-like protein (UBL) and a C-terminal Citrine. D, In-vivo stability of protease substrates in S. cerevisiae. Indicated protease substrates were over-expressed in a S. cerevisiae strain constitutively expressing H2B-CFP. Total cell lysates were analyzed by Western blot with antibodies recognizing the ZZ-tag (upper panel) or Citrine and CFP (middle panel). Equal loading was confirmed by staining the membrane after blotting (lower panel). Bands marked with an asterisk (*) originate from ZZ-tagged proteins cross-reacting with the anti-Citrine/CFP antibody. For complete original blots and stained membranes see S11 Fig. E, Cleavage of UBL substrates in extracts and in S. cerevisiae. ++, highly efficient cleavage; +, cleavage;–, traces cleaved;––, no cleavage; n.d.: not determined; 1, data not shown.

Mentions: The unexpectedly high resistance of xLC3B towards cleavage by Atg4-like proteases originating from other species encouraged us to address the stability of xLC3B and xGATE16 fusions in various eukaryotic cell extracts (Figs 9A and 9B). For control purposes, we also included analogous fusions to trAtg8, scSUMO and the cleavage-resistant scSUMO variant SUMOstar [35,36]. As expected, in wheat germ extract 1 μM of xLC3B- or xGATE16-containing substrate proteins were not significantly processed within 2 h at 25°C, while the corresponding trAtg8 fusion was completely cleaved. In comparison, all substrate proteins harboring Atg8 homologs were completely cleaved both in Xenopus egg extract and rabbit reticulocyte lysate. Interestingly, the scSUMO fusion was only partially cleaved in wheat germ extract and remained stable in rabbit reticulocyte lysate. Control incubations containing a protease mix (0.1 μM each of scUlp1, SUMOstar protease, xAtg4B14-384 and trAtg4) confirmed that the extracts did not contain any substances inhibiting specific proteolytic substrate processing.


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)

Stability of UBL fusions in eukaryotic lysates and in S. cerevisiae.A, Schematic representation of substrates used for (B). B, Stability of protease substrates in cell extracts. Note that in wheat germ extract no proteolytic fragments originating from SUMOstar-, xLC3B- or xGATE16-containing substrates can be detected. For complete blots and stained membranes see S10 Fig. C, Schematic representation of substrates used for expression in S. cerevisiae (D) harboring an N-terminal ZZ-tag, an ubiquitin-like protein (UBL) and a C-terminal Citrine. D, In-vivo stability of protease substrates in S. cerevisiae. Indicated protease substrates were over-expressed in a S. cerevisiae strain constitutively expressing H2B-CFP. Total cell lysates were analyzed by Western blot with antibodies recognizing the ZZ-tag (upper panel) or Citrine and CFP (middle panel). Equal loading was confirmed by staining the membrane after blotting (lower panel). Bands marked with an asterisk (*) originate from ZZ-tagged proteins cross-reacting with the anti-Citrine/CFP antibody. For complete original blots and stained membranes see S11 Fig. E, Cleavage of UBL substrates in extracts and in S. cerevisiae. ++, highly efficient cleavage; +, cleavage;–, traces cleaved;––, no cleavage; n.d.: not determined; 1, data not shown.
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pone.0125099.g009: Stability of UBL fusions in eukaryotic lysates and in S. cerevisiae.A, Schematic representation of substrates used for (B). B, Stability of protease substrates in cell extracts. Note that in wheat germ extract no proteolytic fragments originating from SUMOstar-, xLC3B- or xGATE16-containing substrates can be detected. For complete blots and stained membranes see S10 Fig. C, Schematic representation of substrates used for expression in S. cerevisiae (D) harboring an N-terminal ZZ-tag, an ubiquitin-like protein (UBL) and a C-terminal Citrine. D, In-vivo stability of protease substrates in S. cerevisiae. Indicated protease substrates were over-expressed in a S. cerevisiae strain constitutively expressing H2B-CFP. Total cell lysates were analyzed by Western blot with antibodies recognizing the ZZ-tag (upper panel) or Citrine and CFP (middle panel). Equal loading was confirmed by staining the membrane after blotting (lower panel). Bands marked with an asterisk (*) originate from ZZ-tagged proteins cross-reacting with the anti-Citrine/CFP antibody. For complete original blots and stained membranes see S11 Fig. E, Cleavage of UBL substrates in extracts and in S. cerevisiae. ++, highly efficient cleavage; +, cleavage;–, traces cleaved;––, no cleavage; n.d.: not determined; 1, data not shown.
Mentions: The unexpectedly high resistance of xLC3B towards cleavage by Atg4-like proteases originating from other species encouraged us to address the stability of xLC3B and xGATE16 fusions in various eukaryotic cell extracts (Figs 9A and 9B). For control purposes, we also included analogous fusions to trAtg8, scSUMO and the cleavage-resistant scSUMO variant SUMOstar [35,36]. As expected, in wheat germ extract 1 μM of xLC3B- or xGATE16-containing substrate proteins were not significantly processed within 2 h at 25°C, while the corresponding trAtg8 fusion was completely cleaved. In comparison, all substrate proteins harboring Atg8 homologs were completely cleaved both in Xenopus egg extract and rabbit reticulocyte lysate. Interestingly, the scSUMO fusion was only partially cleaved in wheat germ extract and remained stable in rabbit reticulocyte lysate. Control incubations containing a protease mix (0.1 μM each of scUlp1, SUMOstar protease, xAtg4B14-384 and trAtg4) confirmed that the extracts did not contain any substances inhibiting specific proteolytic substrate processing.

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