Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System

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Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and β isoforms to study their interaction with amyloid β peptide (Aβ42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/β and immobilized Aβ42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.


Enheter & grupper
Externa organisationer
  • University College Dublin

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Biokemi och molekylärbiologi


Sidor (från-till)385-396
Antal sidor12
TidskriftMethods in molecular biology (Clifton, N.J.)
StatusPublished - 2020
Peer review utfördJa