Methods of Protein Purification

1. Methods of Protein Purification Rachel Britt Cox Lab Biochemistry Addition, Rm 337 britt@wisc.edu Biochemistry 660 September 9, 2009

2. Why purify a protein? • Characterize function, activity, structure • Use in assays • Raise antibodies • many other reasons ...

3. Why purify E. coli DinI? • Damage Inducible Protein I • Recombination mediator • Mutant proteins especially difficult to purify • New purification strategy discussed throughout lecture Ala 45 Arg 43 Glu 4 Ser 51

4. Guidelines for protein purification • Define objectives • Define properties of target protein and critical contaminants • Minimize the number of steps • Use a different technique at each step • Develop analytical assays Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

5. How pure should my protein be?

6. How pure should DinI be?

7. Separation of proteins based on physical and chemical properties • Solubility • Binding interactions • Surface-exposed hydrophobic residues • Charged surface residues • Isoelectric Point • Size and shape

8. Basic scheme of protein purification From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

9. Protein preparation, extraction, clarification Cell growth, protein over-expression Cell lysis Removal of cell debris

10. Why use heterologous expression? • Proteins with low natural abundance • Proteins predicted by reverse genetics • Site-directed mutagenesis • Protein engineering

11. Expression systems • Bacteria • Escherichia coli • Lactococcus lactis • other bacteria • Yeast • Pichia pastoris • Pichia methanolica • Saccharomyces cerevisiae • Insect cells • baculovirus • Mammalian cells • Cell Free • wheat germ extract • Escherichia coli extract

12. Expression System Characteristics

13. Expression system for DinI?

14. Expression of DinI in E. coli Plasmid with dinI DinI expression transformation E. coli

15. Inducible promoter system repressor promoter operator gene to be expressed induction promoter operator gene to be expressed RNA Pol promoter operator gene to be expressed

16. Inducible promoter systems

17. Expression of DinI?

18. Expression of DinI T7RNA pol . . . plasmid plasmid . . . T7 RNA pol promoter dinI Translation of dinI mRNA No IPTG + IPTG MW DinI DinI !

19. Protein isolation, concentration, and stabilization Reversible precipitation with salt or organic molecules

20. Fractional precipitation of proteins Discard pellet Precipitate contaminants Add Precipitant, Centrifugation, Discard supernatant, Resuspend protein Add Precipitant, Centrifugation Chromatography Precipitate protein of interest Discard supernatant, Resuspend protein

21. Precipitation of proteins by “salting out” The ability of a salt to precipitate proteins is described by the Hofmeister series: Anions: SCN_ < ClO4_ < NO3_ < Br_ < Cl_ < acetate_ < SO42_ < PO43_ Cations: Na+ < K+ < NH4+

22. Precipitation of proteins with organic polymers Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

23. Fractional precipitation of DinI?

24. Intermediate Purification Liquid chromatography (lower resolution, lower cost)

25. An introduction to liquid chromatography • Protein solution applied to a column • Column = solid porous matrix (stationary phase) + liquid (mobile phase) • Proteins separated based on differing interactions with stationary and mobile phases • Mobile phase conditions can be adjusted to increase or decrease affinity of protein for stationary phase (gradient)

26. Fractionation during chromatography Proteins separated by chromatography are collected in fractions to keep them separated

27. Equipment for liquid chromatography ÄKTA FPLC • Refrigeration • Buffer reservoirs • Gradient maker • Way to apply buffers and protein sample to column • Column • Detection system • Fraction collector • Controller / Recorder

28. Controller / recorder for liquid chromatography

29. Sequence of a general chromatography run

30. Changing buffer between chromatography steps: Dialysis • Need porous membrane with specific molecular weight cutoff (MWCO) • Proteins stay inside membrane • Molecules smaller than MWCO free to equilibrate across membrane • Generally consists of 3, 2-hour steps • Rule of thumb: volume of solution to change into > 100x volume of protein solution http://matcmadison.edu/biotech/resources/proteins/labManual/chapter_4/section4_3.htm

31. Types of liquid chromatography Adsorption Chromatography • Proteins bind to stationary phase • Proteins eluted by altering mobile phase • Includes: affinity, hydrophobic interaction, ion exchange, and chromatofocusing Solution Phase Chromatography • Proteins do not bind to stationary phase • Progress of proteins through column impeded by matrix of stationary phase • Includes: size exclusion chromatography (aka gel filtration)

32. Size exclusion chromatography Smaller Proteins Bigger Proteins

33. Affinity Chromatography

34. Affinity Chromatography • Most commonly-used adsorption chromatography technique • Can be used on protein with natural ligands • Often involves covalent attachment of affinity tag to protein • Because of unique tag, provides rapid, specific cleanup in one chromatography step* • Can allow for automation of protein purification

35. Popular Small Affinity Tags

36. Popular Large Affinity Tags

37. Which Tag to Use? • Specificity of binding interaction • Cost of resin • Native vs. denaturing elution • Presence of metals • Expression level, solubility & toxicity of target protein • Tag removal

38. Tag Removal protein NH2– tag linker considerations: effect on structure effect on function flexibility protein 1° sequence DDDDK protease

39. Tag Removal

40. Which tag for DinI?

41. DinI Nickel Affinity purification of DinI DinI eluted with gradient of imidazole Adapted From:

42. Affinity purification of DinI Increasing [imidazole] Flowthrough MW DinI DinI !

43. Types of liquid chromatography

44. Liquid chromatography and DinI

45. Anion Exchange and DinI UV Q-sepharose column Equilibrate & load in Tris buffer + no salt Elute with linear gradient to 1 M KCl % buffer w/ 1M KCl Conductivity Increasing [KCl] in % of total buffer Increasing Abs @ 280 nm Increasing [Salt] Increasing Volume and Fraction #

46. Anion Exchange and DinI Fraction Samples UV Load MW X1 X2 B12 B8 B10 B6 B4 B2 C7 C9 C12 % buffer w/ 1M KCl Conductivity Pooled Fractions Increasing [KCl] in % of total buffer Increasing Abs @ 280 nm Increasing [Salt] Increasing Volume and Fraction #

47. Polishing steps Liquid chromatography (higher resolution, higher cost) From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

48. Size Exclusion and DinI Conductivity Sephacryl S-100 Load in Tris buffer + 200 mM KCl Elute with Tris buffer + 200 mM KCl UV % buffer w/ 200 mM KCl Increasing [KCl] in % of total buffer Increasing Abs @ 280 nm Increasing [Salt] Increasing Volume and Fraction #

49. Size Exclusion and DinI Fraction Samples Conductivity Load MW G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 H12 UV % buffer w/ 200 mM KCl Pooled Fractions Increasing [KCl] in % of total buffer Increasing Abs @ 280 nm Increasing [Salt] Increasing Volume and Fraction #

50. Liquid chromatography techniques advantages and disadvantages