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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 cleavage characteristics of xAtg4B14-384.A, Time course. Substrates (100 μM) were incubated at 0°C with 500 nM of xAtg4B14-384. At indicated time points, aliquots were withdrawn. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). For a side-by side comparison of selected protease fragments see S4 Fig. B, Temperature dependence of substrate cleavage. 100 μM of xLC3B-MBP (left) or xGATE16-MBP (right) were incubated with xAtg4B14-384 for 1 h at defined temperatures. Note that in comparison to the xGATE16-MBP substrate, twice as much protease was used for cleavage of the xLC3B-MBP substrate. For a comparison of selected protease fragments see S8 Fig. C, Salt sensitivity. 100μM of each substrate was incubated for one hour at 0°C with 500 nM xAtg4B14-384 at NaCl concentrations ranging from 0.2 to 1.5 M. For a comparison of selected protease fragments see S8 Fig. D, P1' preference. Protease substrates used to analyze the P1' preference of xAtg4B14-384 followed the general outline shown in Fig 2A. Here, however, the P1' position of the P1-P1' scissile bond had been mutated to the potentially non-preferred residues methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). Solution cleavage assays were performed with indicated concentrations of xAtg4B14-384 for 1 h at 0°C. Bands marked with an asterisk (*) refer to the protease.
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pone.0125099.g006: In-vitro cleavage characteristics of xAtg4B14-384.A, Time course. Substrates (100 μM) were incubated at 0°C with 500 nM of xAtg4B14-384. At indicated time points, aliquots were withdrawn. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). For a side-by side comparison of selected protease fragments see S4 Fig. B, Temperature dependence of substrate cleavage. 100 μM of xLC3B-MBP (left) or xGATE16-MBP (right) were incubated with xAtg4B14-384 for 1 h at defined temperatures. Note that in comparison to the xGATE16-MBP substrate, twice as much protease was used for cleavage of the xLC3B-MBP substrate. For a comparison of selected protease fragments see S8 Fig. C, Salt sensitivity. 100μM of each substrate was incubated for one hour at 0°C with 500 nM xAtg4B14-384 at NaCl concentrations ranging from 0.2 to 1.5 M. For a comparison of selected protease fragments see S8 Fig. D, P1' preference. Protease substrates used to analyze the P1' preference of xAtg4B14-384 followed the general outline shown in Fig 2A. Here, however, the P1' position of the P1-P1' scissile bond had been mutated to the potentially non-preferred residues methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). Solution cleavage assays were performed with indicated concentrations of xAtg4B14-384 for 1 h at 0°C. Bands marked with an asterisk (*) refer to the protease.

Mentions: We first performed a time course at 0°C with 0.5 μM protease and 100 μM substrate to determine the minimal time required for substrate cleavage (Fig 6A). Fully consistent with earlier results (Fig 2C), near complete cleavage of the xLC3B substrate occurred within 60 minutes. Cleavage of the xGATE16 substrate was even ≈4-fold faster, indicating that complete substrate cleavage is possible within a very short time frame using low protease concentrations and mild cleavage conditions.


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 cleavage characteristics of xAtg4B14-384.A, Time course. Substrates (100 μM) were incubated at 0°C with 500 nM of xAtg4B14-384. At indicated time points, aliquots were withdrawn. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). For a side-by side comparison of selected protease fragments see S4 Fig. B, Temperature dependence of substrate cleavage. 100 μM of xLC3B-MBP (left) or xGATE16-MBP (right) were incubated with xAtg4B14-384 for 1 h at defined temperatures. Note that in comparison to the xGATE16-MBP substrate, twice as much protease was used for cleavage of the xLC3B-MBP substrate. For a comparison of selected protease fragments see S8 Fig. C, Salt sensitivity. 100μM of each substrate was incubated for one hour at 0°C with 500 nM xAtg4B14-384 at NaCl concentrations ranging from 0.2 to 1.5 M. For a comparison of selected protease fragments see S8 Fig. D, P1' preference. Protease substrates used to analyze the P1' preference of xAtg4B14-384 followed the general outline shown in Fig 2A. Here, however, the P1' position of the P1-P1' scissile bond had been mutated to the potentially non-preferred residues methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). Solution cleavage assays were performed with indicated concentrations of xAtg4B14-384 for 1 h at 0°C. Bands marked with an asterisk (*) refer to the protease.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0125099.g006: In-vitro cleavage characteristics of xAtg4B14-384.A, Time course. Substrates (100 μM) were incubated at 0°C with 500 nM of xAtg4B14-384. At indicated time points, aliquots were withdrawn. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). For a side-by side comparison of selected protease fragments see S4 Fig. B, Temperature dependence of substrate cleavage. 100 μM of xLC3B-MBP (left) or xGATE16-MBP (right) were incubated with xAtg4B14-384 for 1 h at defined temperatures. Note that in comparison to the xGATE16-MBP substrate, twice as much protease was used for cleavage of the xLC3B-MBP substrate. For a comparison of selected protease fragments see S8 Fig. C, Salt sensitivity. 100μM of each substrate was incubated for one hour at 0°C with 500 nM xAtg4B14-384 at NaCl concentrations ranging from 0.2 to 1.5 M. For a comparison of selected protease fragments see S8 Fig. D, P1' preference. Protease substrates used to analyze the P1' preference of xAtg4B14-384 followed the general outline shown in Fig 2A. Here, however, the P1' position of the P1-P1' scissile bond had been mutated to the potentially non-preferred residues methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). Solution cleavage assays were performed with indicated concentrations of xAtg4B14-384 for 1 h at 0°C. Bands marked with an asterisk (*) refer to the protease.
Mentions: We first performed a time course at 0°C with 0.5 μM protease and 100 μM substrate to determine the minimal time required for substrate cleavage (Fig 6A). Fully consistent with earlier results (Fig 2C), near complete cleavage of the xLC3B substrate occurred within 60 minutes. Cleavage of the xGATE16 substrate was even ≈4-fold faster, indicating that complete substrate cleavage is possible within a very short time frame using low protease concentrations and mild cleavage conditions.

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