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

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

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

5. Chromatography Sample containing proteins or peptides Liquid flow Liquid flow Separation according to: -molecular weight/ size -charge -hydrophobicity -affinity Time 1 2 3 4 5 4:37 990909

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

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. Always Limit the Number of Steps Maximize the Yield at Each Step Yield (%) 100 80 95% / step 60 90% / step 40 85% / step 20 20% overall yield! 80% / step 75% / step 0 3 5 6 8 1 2 4 7 Number of steps

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”] Vo = “void volume” Vt = “bed volume” Ve = “elution volume” Vi = Vt - Vo

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. Linking Chromatography Techniques Start conditions Technique End conditions GF Small sample volume Diluted sample Buffer change (if required) IEX Low ionic strength High ionic strength orpH change HIC High ionic strength Low ionic strength AC Specific binding conditions Specific elution conditions

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 - electrode Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 + electrode

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