Efficient production of a mature and functional gamma secretase protease

Imran Khan*, Sudarsan Krishnaswamy, Miheer Sabale, David Groth, Linda Wijaya, Michael Morici, Imre Berger, Christiane Schaffitzel, Paul E. Fraser, Ralph N. Martins, Giuseppe Verdile

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

3 Citations (Scopus)
233 Downloads (Pure)

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 (γ)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-β (Aβ) protein. A MultiBac vector containing all components of the γ-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived γ-secretase substrates and proteolysis could be inhibited with γ-secretase inhibitors, confirming specificity of the recombinant γ-secretase enzyme. Finally, affinity purification was used to purify an active recombinant γ-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active γ-secretase complex and provides a new tool to study γ-secretase enzyme and its variants.

Original languageEnglish
Article number12834
Number of pages15
JournalScientific Reports
Volume8
DOIs
Publication statusPublished - 27 Aug 2018

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