The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts.
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.
Abteilung Zelluläre Logistik, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.
Steffen
S
Frey
Abteilung Zelluläre Logistik, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany.
Dirk
D
Görlich
D000818:Animals; D001343:Autophagy / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000235:genetics; D010714:Phosphatidylethanolamines / Q000378:metabolism; D059748:Proteolysis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D014908:Triticum / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; D025801:Ubiquitin; C483858:phosphatidylethanolamine; D010450:Endopeptidases; C099488:TEV protease; D003546:Cysteine Endopeptidases