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

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

Abstract

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.

Detaljer

Författare
Enheter & grupper
Externa organisationer
  • University College Dublin
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Biokemi och molekylärbiologi

Nyckelord

Originalspråkengelska
Sidor (från-till)385-396
Antal sidor12
TidskriftMethods in molecular biology (Clifton, N.J.)
Volym2095
StatusPublished - 2020
PublikationskategoriForskning
Peer review utfördJa