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

McGauran, Gavin ; Linse, Sara LU and O'Connell, David J. (2020) In Methods in molecular biology (Clifton, N.J.) 2095. p.385-396
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... (More)

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.

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author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Biophysical, Calcium, EF hand, Fragment complementation, Glycogen synthase kinase, Human embryonic kidney cells, Single step purification, Surface plasmon resonance
in
Methods in molecular biology (Clifton, N.J.)
volume
2095
pages
12 pages
publisher
Springer
external identifiers
  • scopus:85077172014
  • pmid:31858480
ISSN
1940-6029
DOI
10.1007/978-1-0716-0191-4_22
language
English
LU publication?
yes
id
da2919d4-9e3d-4528-8c04-87aaa82cedfd
date added to LUP
2020-01-10 11:59:49
date last changed
2021-02-23 01:27:00
@article{da2919d4-9e3d-4528-8c04-87aaa82cedfd,
  abstract     = {<p>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.</p>},
  author       = {McGauran, Gavin and Linse, Sara and O'Connell, David J.},
  issn         = {1940-6029},
  language     = {eng},
  pages        = {385--396},
  publisher    = {Springer},
  series       = {Methods in molecular biology (Clifton, N.J.)},
  title        = {Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System},
  url          = {http://dx.doi.org/10.1007/978-1-0716-0191-4_22},
  doi          = {10.1007/978-1-0716-0191-4_22},
  volume       = {2095},
  year         = {2020},
}