1. Saida Almashharawi 2015-2016 Basic Biochemistry CLS 233 Ch4: Protein Purification 1

2. Levels of Structure in Protein Primary: A description of all covalent bonds. The sequence of AA residues Secondary: particularly stable arrangements of AA giving rise to recurring structural patterns. Tertiary: All aspects of the 3D folding of a polypeptide. Quaternary: The spatial arrangement of multisubunits protein 2

3. Protein Purification Crude extract: breaking cells, by osmosis lysis or homogenization. Fractionation: separate proteins into different fraction based on size of charge. Salting out: The solubility of proteins is lowered at high salt concentration. Ammonium sulfate ((NH 4 ) 2 SO 4 ). Dialysis is a procedure to separate proteins from solvents 3

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 4

5. How pure should my protein be؟ ApplicationRequired Purity Therapeutic use, in vivo studies Extremely high > 99% Biochemical assays, X-ray crystallography High 95-99% N-terminal sequencing, antigen for antibody production, NMR Moderately high < 95% 5

6. Separation of proteins based on physical and chemical properties Solubility Binding interactions Surface-exposed hydrophobic residues Charged surface residues Isoelectric Point Size and shape 6

7. Basic scheme of protein purification From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC 7

8. Protein preparation, extraction, clarification Cell growth, protein over- expression Cell lysis Removal of cell debris 8

9. Expression of Target Protein in E. coli DinI expression Plasmid with dinI transformation E. coli 9

10. Protein isolation, concentration, and stabilization Reversible precipitation with salt or organic molecules 10

11. Fractional precipitation of proteins Add Precipitant, Centrifugation Chromatography Precipitate contaminants Precipitate protein of interest Discard supernatant, Resuspend protein Discard pellet Add Precipitant, Centrifugation, Discard supernatant, Resuspend protein 11

12. Intermediate Purification Liquid chromatography (lower resolution, lower cost) 12

13. 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) 13

14. Types of liquid chromatography Type Adsorption or Solution Resin Chemical Group Separation By Equilibrate With Elution With Names of Resins AffinityAdsorptionSpecific LigandLigand Binding Low [Ligand] High [Ligand] Metal, Ig, Hydroxyapatite, Heparin Sepharose, Any ligand Hydrophobic Interaction Adsorption Hydrophobic Groups Hydrophobic Effect High SaltLow Salt Butyl sepharose, Octyl sepharose, Phenyl sepharose Anion ExchangeAdsorptionPositively charged ions Coulombic Interacions Low SaltHigh Salt Mono-Q, Source-Q, DEAE Cation ExchangeAdsorptionNegatively charged ions Coulombic Interacions Low SaltHigh Salt Mono-S, Source-S, CM ChromatofocusingAdsorptionNegatively charged ions Isoelectric PointPoly-bufferpH gradient Mono-P Size Exclusion (gel filtration( Solution Phase PoresSize / Shape of Protein Same Buffer Sephacryl #, Sephadex # 14

15. Polishing steps From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC Liquid chromatography (higher resolution, higher cost) 15

16. Liquid chromatography techniques advantages and disadvantages Type of Chromatography AdvantagesDisadvantagesResolution Affinity Quick and specific Resins and ligands can be expensive Low to Medium Hydrophobic Interaction Can be used directly from ammonium sulfate precipitation Relatively low resolution and binding capacity Low to Medium Ion Exchange Versatile resin choices Protein solution must start at low [salt] Medium to High Chromatofocusing High resolution pH gradient can be harsh for protein High Size Exclusion Distinct from other techniques, Can be used analytically or for buffer exchange Long run time Low to High 16

17. Protein detection methods SDS-PAGE –Visual confirmation UV Spectrophotometry –Absorbance @ 280 nm –Due mostly to Trp –Protein] calculated with Beer’s Law Colorimetric Techniques –Color change proportional to [protein] –Bradford, Lowry, BCA J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29 17

18. Final steps in purification Check purity by detection methods Test for interfering contaminants –Nucleases –Proteases –Toxins Concentrate your protein –Precipitation –Lypholyzation –Small column with high binding capacity Choose a storage buffer and storage conditions –Consider intended use of protein –Stabilizing additives –Flash freeze protein and store at -80 o C Confirm identity of purified protein –Mass spectrometry –N-terminal sequencing –Analytical assays 18

19. Basic scheme of protein purification Liquid chromatography (higher resolution, higher cost) Liquid chromatography (lower resolution, lower cost) Reversible precipitation with salt or organic molecules Cell growth, protein over- expression Cell lysis Removal of cell debris 19

20. Separation Processes that can be Used to Fractionate Proteins Precipitation Separation Process ammonium sulfate Basis of Separation solubility polyethyleneimine (PEI)charge, size isoelectricsolubility, pI Chromatography gel filtration (SEC(size, shape ion exchange (IEX(charge, charge distribution hydrophobic interaction(HIC) hydrophobicity DNA affinityDNA binding site immunoaffinity (IAC(specific epitope chromatofocusingpI Electrophoresis gel electrophoresis (PAGE(charge, size, shape isoelectric focusing (IEF(pI Centrifugation sucrose gradientsize shape, density Ultrafiltration ultrafiltration (UF(size, shape 20

21. The expansion of the protein band in the mobile phase is caused by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein improves. Rate is decreased and resolution can decline because of the diffusional spreading 21 Protein Purification: - Column Chromatography

22. Ion-exchange Chromatography Cation exchangers contain negatively charged polymer Anion exchangers contain positively charged polymer. Is effected by pH 22

23. Size-Exclusion Chromatography Also calledgel filtration: The column matrix is a cross- linked polymer with pores of selected size. Larger protein migrate faster than smaller ones because they are too large to enter the pores 23

24. Affinity Chromatography Separate protein by their binding specificities. The proteins retainedon the column are those that bind specifically to a ligand cross- linked to the beads. Proteins that do not binds to ligands are washed through to column 24

25. Electrophoresis Separation of porteins is based on the migration of charged protein in an electric field The migration of a protein in a gel during electrophoresis is a function of its size and shape  =V/E=Z/ f  : electrophoretic mobility V: velocity; E: electrical potential Z: net charge; f: frictional coefficient 25

26. PyaGE SDS-PAGE: Sodium Dodecyl Sulfate (SDS) pol acrylamidegellectrophoresis SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus,each protein has a similar charge- to-mass ratio. Coomassie blue stains protein. Western blot 26

27. Estimating the Molecular Weight of a Protein 27

28. Isoelectric Focusing pI of a protein: net charge=0 A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI 28

29. Two-Dimensional Electrophoresis Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW. 29

30. Activity Vs. Specific Activity Unit : amount of enzyme causing transformation of 1  mole of substrate per min. at 25 o C under optimal conditions Activity : Total units of enzyme (U). Specific activity : (U/mg) of total protein 30

31. Assignment #3 31 Q1- What is the principle for each of the following technique: A- Gel filtration B- Ion Exchange chromatography Q2- Separation of proteins based on physical and chemical properties, What are these properties?