Aims: -Purification of a specific protein. -Study of protein-protein interactions

Transcription

1

2 Aims: -Purification of a specific protein -Study of protein-protein interactions

3

4 This is a reliable method for purifying total IgG from crude protein mixtures such as serum. Protein A (linked to resin beads) binds to the Fc portion of IgG (immunoglobulin). The beads are used in a chromatography column or loose in a test tube.

5

6 globalmedicaldiscovery.com

7

8 Pull-down assays: Use strong non-covalent interactions between a protein of interest (bait) and the potential interacting partners (prey proteins).

9 The bait can be expressed in E. coli as a fusion protein. The fusion protein is then immobilized on a solid support or solid particles using an affinity ligand specific for the fusion tag. Other unwanted products from E. coli are washed off. The immobilized bait protein is incubated with the prey protein (in a mixture), and other non-interacting molecules can be rinsed off.

10

11

12

13

14 A common method to generate a fusion protein (bait) is to use GST (glutathione-s transferase) as the fusion tag by expression in E. coli. Fusion at C-terminus of GST. The GST tag interacts very strongly with glutathione (GSH). The fusion protein is then immobilized on agarose particles bearing GSH. Other unwanted products are washed off. The immobilized bait protein is incubated with the prey protein, and non interacting molecules can be rinsed off.

15

16 GSH (γ-glutamylcysteinylglycine)

17 Specific GST approach: membrane-associated proteins

18 Specific GST approach: Use a GST-PDZ fusion protein to pull down membrane associated proteins by affinity from HeLa cells. Expression of GST-PDZ in E. coli and attachment to agarose-glutathione particles; washing. Incubation of particles with HeLa cancer cells to capture interactive proteins. Non interacting proteins rinsed off. Elution of captured proteins and separation by SDS-PAGE. J Proteome Res Sep;5(9):

19

20 Add all proteins from HeLa cells Stir and let equilibrate; centrifuge. Proteins with no affinity for PDZ1 are in the supernatant. Detach proteins of interest from beads using a strong eluent.

21 PDZ affinity proteins on SDS-PAGE ~36 kda Connexin 36?

22 Identification of other proteins pulled down by GST-PDZ Keratin and cytokeratin 8 Trypsin (contaminant) GST-PDZ1 Vimentin Actin Alpha-actinin kda ARN/ADN nuclear binding protein 54 kda Proline/glutamine-rich splicing protein 76 kda Nucleolin 74 kda Beta-tubulin kda Carbonic anhydrase + connexin 36 kda

23 Another common bait used for pulling down proteins: the His 6 -tag The tag may be placed at the N or C terminus: (His) 6 -Protein or Protein-(His) 6

24 His tags have a high affinity for Ni 2+ and Co 2+

25 A mixture of proteins (including the His-tagged fusion protein) is incubated with an affinity resin containing chelated Ni 2+ or Co 2+ Resins are available commercially in different varieties, and are generally sepharose/agarose functionalized with a chelator. Immobilized metal affinity chromatography (IMAC)

26 Chelators: iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) are used for Ni 2+

27 Carboxylmethyl aspartate (Co-CMA) is used for cobalt The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with Ni 2+ or Co 2+ Imidazole in high conc. (200 mm) is then used to detach His-tag proteins.

28 Affinity chromatography is aimed at protein purification, although it can yield the protein of interest plus interacting partners. As opposed to chromatography, affinity pulldown methods are often aimed at studying protein-protein interactions rather than purification. GST tags are quite bulky and can significantly change the properties of the bait protein to which they are fused. His tags are much smaller and in most cases do not change the bait protein s properties. Affinity and pulldown methods are usually followed by SDS- PAGE.