1. Biochemistry 412 2004 20 February Lecture Analytical & Preparative Protein Chemistry II

2. Positively-charged basic residues (K, R, & H) Negatively-charged acidic residues (E & D) Hydrophobic “patch” Ligand binding pocket (active site) ca. 40 Å Macromolecular dimensions: Proteins are Amphiphilic Macro-Ions >>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior.

5. 5 Chromatography Liquid flow Liquid flow 4:37 990909 Time12345 Separation according to: -molecular weight/ size -charge -hydrophobicity -affinity Sample containing proteins or peptides

7. 7 Purity Step Capture Intermediate purification Polishing Isolate product, concentrate, stabilize Remove bulk impurities Achieve final purity. Remove trace impurities, structural variants, aggregates, viruses, etc. Three Phase Strategy: An aid in developing the purification scheme

8. 8 Sample Preparation General considerations: Select extraction procedure according to source and location of protein Use gentle procedures to minimize acidification and release of proteolytic enzymes Work quickly at sub-ambient temperatures Use buffer to maintain pH, ionic strength Goal: To stabilize sample

9. 9 Always Limit the Number of Steps Maximize the Yield at Each Step Number of steps Yield (%) 95% / step 90% / step 85% / step 80% / step 75% / step 0 20 40 60 80 100 12 3 4 56 7 8 20% overall yield!

10. Gel Filtration

11. Gel Filtration (GF) Chromatography

12. The principle of gel filtration -- excluded volume [Note: gel filtration chromatography is also sometimes called “size exclusion chromatography”] V o = “void volume” V t = “bed volume” V e = “elution volume” V i = V t - V o

13. Principles of gel chromatography (con’d)

14. Gel Filtration Elution Volumes as a Function of Molecular Weight Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

15. Ion Exchange Chromatography

16. Ion Exchange (IEX) Chromatography

17. Ion Exchange Chromatography (con’d)

18. Some other popular chromatographic methods: Hydrophobic interaction chromatography Affinity chromatography Reverse phase chromatography

19. Hydrophobic Interaction Chromatography (HIC)

20. Affinity Chromatography

21. “Reversed Phase” Chromatography (RPC) (elution with organic solvents)

23. 23 TechniqueEnd conditionsStart conditions Small sample volume GF Diluted sample Buffer change (if required) Low ionic strength IEX High ionic strength or pH change High ionic strength HIC Low ionic strength Specific binding conditions AC Specific elution conditions Linking Chromatography Techniques

24. In addition, there are non-chromatographic protein purification techniques, e. g.: Ammonium sulfate precipitation Sedimentation (rare) Recombinant gene product over-expression Refractile body prep (see above) Detergent extraction Heat treatment (especially for recombinant thermophile proteins expressed in E. coli) Etc.

25. Once You’ve Purified Your Protein, How Do You Characterize It? Some typical analytical tests: - SDS PAGE (both reducing and non-reducing) - Bioassay (if you have one) - Total protein determination - UV spectrophotometry - CD spectrometry - disulfides? - amino acid analysis - N-terminal (& C-terminal?) sequencing - HPLC? - metal analysis - mass spectrometry - NMR spectrometry & X-ray crystallography* - other? *Not usually used for routine analytical purposes!!

26. Protein Size Determination by SDS Polyacrylamide Gel Electrophoresis Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 + electrode - electrode

27. Circular Dichroism Spectroscopy of Polypeptides Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984