Imran Khan, Edith Cowan UniversityFollow
Sudarsan Krishnaswamy, Edith Cowan UniversityFollow
David M. Groth
Linda Wijaya, Edith Cowan UniversityFollow
Michael Morici, Edith Cowan UniversityFollow
Paul E. Fraser
Ralph N. Martins, Edith Cowan UniversityFollow
Nature Publishing Group
Centre of Excellence for Alzheimer’s Disease Research and Care / School of Medical and Health Sciences
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.
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