Efficient production of a mature and functional gamma secretase protease.

ABSTRACT: Baculoviral protein expression in insect cells has been previously used to generate large quantities of a protein of interest for subsequent use in biochemical and structural analyses. The MultiBac baculovirus protein expression system has enabled, the use of a single baculovirus to reconstitute a protein complex of interest, resulting in a larger protein yield. Using this system, we aimed to reconstruct the gamma (?)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-? (A?) protein. A MultiBac vector containing all components of the ?-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived ?-secretase substrates and proteolysis could be inhibited with ?-secretase inhibitors, confirming specificity of the recombinant ?-secretase enzyme. Finally, affinity purification was used to purify an active recombinant ?-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active ?-secretase complex and provides a new tool to study ?-secretase enzyme and its variants.

SUBMITTER: Khan I 

PROVIDER: S-EPMC6110731 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature