1. CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Protein purification and characterization

2. CHMI 2227 - E.R. Gauthier, Ph.D.2 Protein purification

3. CHMI 2227 - E.R. Gauthier, Ph.D.3 Protein purification General procedure Homogenization Crude protein extract Coarse purification steps Chromatography 1, 2, 3….x steps PURE! (well, let’s hope so…) Monitor presence of protein of interest: - Activity - Purity - Quantity

4. CHMI 2227 - E.R. Gauthier, Ph.D.4 Protein purification 1. Coarse methods

5. CHMI 2227 - E.R. Gauthier, Ph.D.5 Differential precipitation 1.1. Precipitation by adjusting the pH pH solubility pI

6. CHMI 2227 - E.R. Gauthier, Ph.D.6 Differential precipitation 1.2.Salting out http://www.tulane.edu/~biochem/med/pure.htm

7. CHMI 2227 - E.R. Gauthier, Ph.D.7 Differential precipitation 1.2.Salting out Mixture of proteins in buffer: A: precipitates at salt [ ] = 15% B: precipitates at salt [ ] = 25 % C: precipitates at salt [ ] = 35% 1)Add (slowly) (NH 4 ) 2 SO 4 to 20% 2)Centrifuge to pellet precipitated proteins Pellet: protein A Supernatant: protein B + C 1)Add (slowly) (NH 4 ) 2 SO 4 to 30% 2)Centrifuge to pellet precipitated proteins Pellet: protein B Supernatant: protein C

8. CHMI 2227 - E.R. Gauthier, Ph.D.8 Dialysis Hours

9. CHMI 2227 - E.R. Gauthier, Ph.D.9 Centrifugation

10. CHMI 2227 - E.R. Gauthier, Ph.D.10 Protein purification 2. Fine methods Column Fraction collector Buffer reservoir Pumps Sample injector

11. CHMI 2227 - E.R. Gauthier, Ph.D.11 Protein purification 2.1. Ion exchange chromatography

12. CHMI 2227 - E.R. Gauthier, Ph.D.12 Protein purification 2.1. Ion exchange chromatography DesorptionAdsorption Anion exchanger NaCl Example of cation exchange chromatography

13. CHMI 2227 - E.R. Gauthier, Ph.D.13 Protein purification 2.1. Ion exchange chromatography Progressive, linear change in NaCl concentration Stepwise gradient of NaCl concentration Add buffer

14. CHMI 2227 - E.R. Gauthier, Ph.D.14 Protein purification 2.2. Molecular sieve chromatography

15. CHMI 2227 - E.R. Gauthier, Ph.D.15 http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_Prod~SelGuides~Media~GFSelG

16. CHMI 2227 - E.R. Gauthier, Ph.D.16 Protein purification 2.2. Molecular sieve chromatography

17. CHMI 2227 - E.R. Gauthier, Ph.D.17 Protein purification 2.3. Affinity chromatography

18. CHMI 2227 - E.R. Gauthier, Ph.D.18 Analysis of proteins Electrophoresis

19. CHMI 2227 - E.R. Gauthier, Ph.D.19 Electrophoresis 1. SDS-PAGE Electrophoresis SDS

20. CHMI 2227 - E.R. Gauthier, Ph.D.20 Electrophoresis 1. SDS-PAGE Electrophoresis

21. CHMI 2227 - E.R. Gauthier, Ph.D.21 Electrophoresis 1. SDS-PAGE Electrophoresis http://wine1.sb.fsu.edu/bch5425/lect20/lect20.htm www.biology.ucsd.edu/classes/bggn224.FA06/crash_2.ppt

22. CHMI 2227 - E.R. Gauthier, Ph.D.22 Electrophoresis 1. SDS-PAGE Electrophoresis

23. CHMI 2227 - E.R. Gauthier, Ph.D.23 Electrophoresis 1. SDS-PAGE Electrophoresis Log Mr Distance migrated from well (cm)

24. CHMI 2227 - E.R. Gauthier, Ph.D.24 Electrophoresis 2. Isoelectrofocusing

25. CHMI 2227 - E.R. Gauthier, Ph.D.25 Electrophoresis 2. Isoelectrofocusing pH in gel = pI of proteins

26. CHMI 2227 - E.R. Gauthier, Ph.D.26 Electrophoresis 3. Two-dimensional gel electrophoresis

27. CHMI 2227 - E.R. Gauthier, Ph.D.27 Electrophoresis 3. Two-dimensional gel electrophoresis Each spot is a single protein

28. CHMI 2227 - E.R. Gauthier, Ph.D.28 Electrophoresis 4. Western blot analysis Structure of an antibody

29. CHMI 2227 - E.R. Gauthier, Ph.D.29 Electrophoresis 4. Western blot analysis

30. CHMI 2227 - E.R. Gauthier, Ph.D.30 Protein purification Example and data analysis

31. CHMI 2227 - E.R. Gauthier, Ph.D.31 Protein purification Example and data analysis Coomassie-blue-stained PAGE-SDS gel

32. CHMI 2227 - E.R. Gauthier, Ph.D.32 Protein purification Example and data analysis

33. CHMI 2227 - E.R. Gauthier, Ph.D.33 Protein sequencing

34. CHMI 2227 - E.R. Gauthier, Ph.D.34 Protein sequence - Sickle-cell anemia Normal red blood cell Sickled red blood cell

35. CHMI 2227 - E.R. Gauthier, Ph.D.35 Determination of protein sequence 1. Enzyme mapping EnzymeAmino acidCutting site TrypsinArg/LysC-ter ChymotrypsinPhe/Trp/TyrC-ter Protease V8Asp/GluC-ter PepsinPhe/Trp/TyrN-ter ThermolysinLeu/Ile/Trp/Tyr/ Val/Ala/Phe N-ter Carboxypeptidase A All C-ter a.a. except Pro, Arg/Lys - Free amino acids from the C-ter - Doesn’t cut if Pro is the penultimate amino acid Carboxypeptidase B Only Arg/Lys when C-ter Based on the property of some enzymes to cut the peptide bonds next to specific amino acids; ChemicalAmino acid Cutting site Cyanogen bromideMetC-ter  -mercaptoethanol CysDisulfide bonds IodoacetateCysPrevents the reduction of disulfide bonds 1) 1-Fluoro-2,4 dinitrobenzene (FDNB) 2) Dansyl chloride 3) Dabsyl chloride Destroy all the amino acids with the exception of the one at the N-terminus. HydrazineDestroy all the amino acids with the exception of the one at the C-terminus. NOTE: Trypsin, Chymotrypsin, protease V8, pepsin and thermolysin do NOT cut if Pro is part of the peptide bond.

36. CHMI 2227 - E.R. Gauthier, Ph.D.36 Determination of protein sequence 1. Enzyme mapping – example 1

37. CHMI 2227 - E.R. Gauthier, Ph.D.37 Determination of protein sequence 1. Enzyme mapping – example 2  HCl 6M: Ala, Gly 2, Lys, Met, Ser, Thr, Tyr  CNBr: 2 peptides were obtained: Peptide 1: Ala, Gly, Lys, Thr Peptide 2: Gly, Met, Ser, Tyr  Trypsin: 2 peptides were obtained: Peptide 3: Ala, Gly Peptide 4: Gly, Lys, Met, Ser, Thr, Tyr  Chymotrypsin: 2 peptides were obtained: Peptide 5: Gly, Tyr Peptide 6: Ala, Gly, Lys, Met, Ser, Thr  FDNB: yields Gly  Carboxypeptidase A: yields Gly The following data were obtained after treating an octopeptide with the following reagents: What is the sequence of this peptide?

38. CHMI 2227 - E.R. Gauthier, Ph.D.38 Determination of protein sequence 2. Edman degradation

39. CHMI 2227 - E.R. Gauthier, Ph.D.39 Determination of protein sequence 2. Edman degradation Identify

40. CHMI 2227 - E.R. Gauthier, Ph.D.40 Determination of protein sequence 3. Mass spectrometry

41. CHMI 2227 - E.R. Gauthier, Ph.D.41 Determination of protein sequence 3. Mass spectrometry

42. CHMI 2227 - E.R. Gauthier, Ph.D.42 Determination of protein sequence 3. Mass spectrometry