Identification, production and purification

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

5. His-tag

6. Ni-NTA Ni-IDA

8. GST-affinity

9. Strep tag-II Elution with biotin analog: desthiobiotin

10. CBP affinity

12. Comparison of affinity tag technologies Lichty JJ 2005 PEP May 41(1):98-105

13. Sf9 ou H5 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