Diapositive 1

1
Identification, production and purification
Identification methods Purification strategies
Single gene expression and in vitro
assembly Co-expression techniques
2
Choice of the purification strategy
- Most widely used tags His, GST, strep, FLAG
- Use two different affinity tags
3
Tandem affinity purification
Apply the cell lysate to the first affinity
resin Wash unbound proteins and contaminants
Elute tagged protein I and interacting partner
with elution buffer I Apply the cell lysate
to the second affinity resin Wash unbound
proteins and contaminants Elute tagged protein
II and interacting partner with elution buffer II
Affinity resin I
Affinity resin II
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5
His-tag
6
Ni-NTA
Ni-IDA
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8
GST-affinity
9
Strep tag-II
Elution with biotin analog desthiobiotin
10
CBP affinity
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Comparison of affinity tag technologies
Lichty JJ 2005 PEP May 41(1)98-105
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Production of multi-protein complexes
Co-infection
His-tagged subunit Flag-tagged
subunit GST-tagged subunit native subunit
I 2 50 mM
I 2 5mM
FT Talon
I 5mM
IP Flag
Wash
MW
His-p89
p80
His-tag
p62
p44, p52
p34F
Flag
1 litre, 300 ml dépot 20 ml