1. Table 5-1 Protein Purification Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.) Two main aproaches Classical – from the natural source (e.g. tissue) Advantages: natural (modifications, binding partners, etc.) Disadvantages: intractable for low abundance proteins (~20,000 types of proteins in the cell!) Molecular cloning (insert gene into heterologous host and induce cells to make lots of the protein of interest Advantages: abundance, purification tricks, genetic variations easy Disadvantages: may lack natural modifications, partners, etc.

2. Text, Figure 5-2 Overexpressing a protein faster than the host cell (E. coli) can fold it leads to aggregated protein in an inclusion body. After isolating inclusion bodies (by cell disruption and centrifugation), often the desired protein can be unfolded and successfully refolded. [Not necessarily a desirable approach] Protein Overexpression

3. Figure 5-4 Tryptophan has a strong absorbance at 280nm. This is useful for detecting the presence of proteins during chromatography, and in estimating protein concentration. To be accurate, one needs to know the amino aid composition of the protein, especially how many Trp’s there are. This is usually know from genetic data. Keeping track of proteins during purification Text, Figure 5-4

4. Page 97 Properties of proteins that form the basis for various purification strategies Heat stability precipitation at high Temp

5. Purification by ammonium sulfate precipitation Everything has a finite solubility The solubility of a protein tends to decrease as the concentration of salt (especially polyvalent) increases The solubilities of different proteins in ammonium sulfate are different proteins Therefore, as you increase the A.S., different proteins reach their solubility limits and come out of solution (i.e. precipitate) at different points Increasing ammonium sulfate Advantages: Scales-up well Simple, cheap Disadvantages: Not very specific The protein must be at fairly high concentration or its solubility limit may not be reached Text, Figure 5-5

6. Figure 5-6 Ion-exchange chromatography separates proteins mainly based on charge properties Anion exchange: Use a column whose matrix is positively charged (e.g. quaternary ammonium groups) Add protein mixture to column at reasonably high pH (Why?) Elute by running through solution with increasing salt concentration (Why does this work) [Cation exchange chromatography is the reverse] Text, Figure 5-6

7. Figure 5-7 Size exclusion or gel filtration chromatography: Separation based on molecular size (of the native complex). Large molecules migrate fastest(!) Advantages: Informative (e.g.regarding native size) simple Disadvantages: Capacity limited Not very good resolving power (~ factor of 2 in size) Text, Figure 5-7

8. Affinity chromatography: separates proteins based on specific binding property Examples: Attach ligand that the protein binds to the column matrix Attach an antibody to the column that recognizes the protein Use molecular cloning techniques to add a short sequence tag to the protein of interest, and make a chromatography matrix that binds the tag Super powerful, specific General, doesn’t require any special property of the protein Most common: metal (e.g. Ni) attached to column, His 6 tail attached to protein Terminal tail can be cut off at the end Text, Figure 5-8

9. Figure 5-8 Monitoring the progress of purification, and knowing if you’ve got the right protein: Gel electrophoresis

10. Figure 5-9 SDS-PAGE (polyacrylamine gel electrophoresis): Often used for monitoring progress of purification Protein chains are denatured by the SDS detergent The SDS is (-) charged and tends to bind similarly (i.e. proportionally) to all proteins Then, negatively charged electrode in electrophoresis drives proteins downward in the gel (driving force is proportional to protein size, but frictional force is more strongly dependent on size, so large proteins migrate more slowly) A lane containing a ‘ladder’ of molecular weight standards run at the same time, makes it possible to estimate the MW of the protein chains

11. Often used for monitoring progress of purification Protein chains are denatured by the SDS detergent The SDS is (-) charged and tends to bind similarly (i.e. proportionally) to all proteins Then, negatively charged electrode in electrophoresis drives proteins downward in the gel (driving force is proportional to protein size, but frictional force is more strongly dependent on size, so large proteins migrate more slowly A lane containing a ‘ladder’ of molecular weight standards run at the same time, makes it possible to estimate the MW of the protein chains SDS-PAGE (polyacrylamine gel electrophoresis): Text, Figure 5-10

12. Page 96 Coomassie: an example of a protein specific dye that is used widely to detect protein bands in gels

13. Page 96 An example of SDS-PAGE from the lab, showing the progress of protein purification Crude mixture (e.g. whole cells) Single fraction after Ni-column chromatography (and other purification Molecular weight standards kDa

14. Figure 5-11 1.Run one kind of electrophoresis 2.Cut out lane 3. Rotate, and run second type of electrophoresis An example of 2D gel electrophoresis: tremendous resolving power Text, Figure 5-11

15. Using ELISA to detect the presence of a particular protein of interest You would typically run an ELISA experiment (or other activity based assay) on each of the tubes or ‘fractions’ coming off a chromatography column Text, Figure 5-3