Protein Purification and Characterization

1. Protein Purification and Characterization • Why Study proteins? • IMPORTANT FACTORS IN PROTEIN PURIFICATION • Starting materials • tissues, cells or clones expressed in E. Coli or animal cells • Decisions- • quantity of protein, protein modification availability of samples, is it cloned yet, expense

2. Stabilization of protein is key -proteins are not meant to be purified, so you need to keep them alive and happy (active / native) • pH- both activity and structure are pH dependent • Temperature- most stabile at low temperature - reduces energy in the system for unfolding and reduces the protease kinetics. Few proteins are unstable at low temps - ppdk (Dr. Chastain's enzyme) and the ATPase in mitochondria • Protease inhibitors- several classes of proteins catalyze the hydrolysis of peptide bonds (called proteases). Usually need to add several "suicide" inhibitors and reduce free metals which are used by the proteases • Reducing agents - beta - mercaptoethanol and dithiolthreitol both act as reducing agents. Prevent the oxidation of amino acids • Detergents- Membrane bound proteins often need added detergents (soaps) to mimic the ampipathic nature of the membrane you so cruelly ripped it from - need to be above the concentration at which micelles are formed - the critical micellular concentration (CMC)

3. Homogenization- breaking the cell apart Mechanical Shearing - Warring blender, glass or plastic pestle like homogenizer Freeze Thaw - cycles under hypnotic conditions, ice crystals disrupts membranes Enzymatic degradationof the cell membrane - mostly for bacterial preparation Sonication - high energy sounds to disrupt membrane Detergentdisruption of memb

4. METHODS OF PURIFICATION • Centrifugation -Separation based on density, mass, shape and the density of the solution • Sedimentation of particles measured by Svedburg units • Force applied in gravitational (g) forces • The centrifugal force depends on speed and time and radius of rotor • Differential centrifugation • One of the most used methods in biochemistry • Uses increasing g forces to yield a pellet and a supernatant • Subcellular centrifugation - a way to separate the cell contents based on density of organelles • Cytosol - not an organelle but a result of centrifugation

5. Differential centrifugation • Use of density of organells to isolate cell fractions

6. Density gradient centrifugation Also called Zonal centrifugation - Performed in the presence of an increasing dense solution often sucrose or other materials (percol most common) can be used to purify a specific organelle or determine the sedimentation and ultimately the molecular weight of a protein

7. Ammonium sulfate precipitation - salting out proteins • At high concentrations of this strong salt, water is highly ordered High concentration of strong chaotropic salts “strips” water away from protein Lower availability of solvent (water) This favors protein interactions rather than protein - solvent interactions causes aggregation of proteins (they become insoluble) • Each protein has a different solubility so this is a method to isolate groups of protein Precipitation is reversible and usually non damaging to structure of the enzyme Ammonium Sulfate is most commonly used. Urea is also used but is usually is harder for the protein to refold.

8. Column Chromatography • Separation based the interactions between a mobile phase and the chromatographic media (stationary phase) • Used to separate any of the big four biomolecules

9. Column Chromatography • Separation based the interactions between a mobile phase and the chromatographic media (stationary phase) • Used to separate any of the big four biomolecules • Components • Column • Pump • Absorbance monitor • Conductivity monitor • Fraction collector • Controller - for more advanced work (control freaks?)

10. Affinity chromatography purification based on a natural interactions for a protein and a substrate or chemical group (ligand) • only proteins which recognize the molecule on the stationary phase will bind • Elute by competition with the bound ligand • generally a good method but it doesn’t always work - Some non-specific interactions can occur • Spacer arm may be needed to make the compound available to the protein

11. Examples of ligand - protein affinity matrix • ATP. Glutathione, nickel – small molecules attached to a ligand • Fusion proteins can take advantage of affinity by acting as a tag: • glutathione S transferase (GST) –binds to glutathione • histidine6 - binds to a nickel column • Power of biochemistry and molecular biology an example of affinity chromatography • Ras - small protein involved in several cancers • Low concentration in cells, so it difficult to purify and study • create a fusion protein 1/2 Ras 1/2 Glutathione S-transferase (GST) and produce large amounts of it. • lead to discovery of additional proteins involved in Ras regulation

12. Ionexchangechromatography • - separation of proteins based on net charge of protein - exchange of ions for proteins • Anion Exchanger • weak exchanger - diethylaminoethyl (DEAE) • strong exchanger - quatenaryaminoethyl (QAE) • This type of resin is positively charged • The resin binds negative proteins • Proteins are eluted by NaCl or altering pH - how does this work?

13. Cation exchanger • weak exchanger - carboxymethy (CM) • weak exchanger - sulfipropyl (SP) • protein eluted by the same means as above

14. Size exclusion (SEC) or gel filtration chromatography Media (solid phase) is a defined pore sizes in polymer beads, large molecules “go around” small molecules “go through and around the beads” Smaller sized proteins are retained and come out last Range of types of beads and chemistry - resin can be made of agarose, acrylamide or other polymers

15. Size exclusion (SEC) or gel filtration chromatography • Can be used to separate proteins, remove salts exchange buffers or to determine the molecular weight of a purified protein • Also used to determine the molecular weight of a protein - use protein standards with known molecular weights, prepare a standard curve of these known proteins and compare the elution volumes of the knowns to the unknowns

16. Example • Sephacryl S-200 has a fractionation range of 5 kDa to 250 kDa What is the exclusion limit? • Would this be appropriate for a set of proteins with molecular weights of 8 kDa, 15 kDa, 200 kDa and 500 kDa? • What about 15, 250, 310, 405 kDa • if you wanted the 15 kDa protein? • What about if you wanted to purify the 310 kDa protein?

17. Protein Characterization • Electrophoresis - The transport of particles by an electrical field through a solid media • - a good method for determining the purity of a protein and analyze a mixture of proteins • - Separation of charged compounds based on an applied electrical field, net charge and frictional coefficient (mass and shape of molecule) • Similar to DNA gels - proteins and very small DNA (oligonucleotides) use acrylamide

18. PAGE (Polyacrylamide Gel Electrophoresis) the Gel is a polymerized Acrylamide-alter the ratio and concentration of polymer and crosslinker to alter the pore size and change the • migration through the gel • - A low % gel (acrylamide) will separate higher MW proteins while smaller proteins are not well resolved • -Stacking Gel vs. Resolving Gel - need to get the proteins to start at the same time (compressing the proteins into a • narrow starting zone at the resolving gel)

19. Denatured Electrophoresis - SDS PAGE • Separation of proteins based on size not charge • Add a reducing agent - ß-mercaptoethanol or dithiothreitol - and a detergent - sodium dodecyl sulfate (SDS) - then boil to unravel the protein and solvate protein with ampipathic SDS • - each SDS has 2 negative charges • - many SDS per molecule • - total amount of SDS bound is proportional to the MW • - Each protein molecule will be sufficiently negative • Therefore each protein will be very negativity charge regardless of the amino acid composition, • - The size of protein influences the migration - separation is based on size only not charge.

20. Native Gel Electrophoresis • Separations based on native size and charge • Two proteins of a similar size but different charge will migrate differently • Protein interactions can influence the migration of protein

21. Isoelectric Focusing Electrophoresis • Separation of proteins based on isoelectric point • Native or denatured electrophoresis in a pH gradient of polyampholytes • pH gradient is formed when electrical field is applie • Proteins will migrate, depending on net charge, until there is no longer a charge on the protein. • How does this happen? • 2 Dimensional Electrophoresis • Combination of native or denatured PAGE and IEF • Run in two directions • 1- PAGE - to separate by size • 2- IEF to separate by charge alone • Good to separate very crude mixtures or determine the difference between two proteins that are the same size • but with a different pI

22. 2D-Electrophoresis 2D-electrophoresis allows separation of proteins by both size and isoelectric point. Each spot represents a different protein. The horizontal represents the isoelectric focusing direction, while the veritcal represents the SDS PAGE direction.

23. Immuno Analysis • Immunoglobins - 5 major classes main antibody response in sera is IgG • antigen - foreign substance that triggers antibody formation • epitope - section of antigen that antibody recognizes • Antibodies consist of heavy and light chains • Fab region - highly variable - recognize target (antigen) • FC heavy chain - interacts with other proteins • polyclonal vs. monoclonal antibodies

24. polyclonal • from sera of an animal • several epitopes to the same antigen • some may cross react with other proteins in a non-specific manner • produce lots of antibodies al long as the animal lives and you continue to boost • monoclonal • derived from single cell - hybrid of mouse spleen and a immortal cell line (lymphocyte and myeloma) • inject mice then can grow cell in a dish • antibodies purified from cell culture media • single epitope, very specific • unlimited production of antibodies

25. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) Plastic Dish

26. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 1 Protein of interest is Bound to plastic Plastic Dish

27. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 2 Unreacted binding sites are Covered with a non-reactive protein 1 Protein of interest is Bound to plastic Plastic Dish

28. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 2 Unreacted binding sites are Covered with a non-reactive protein 3 Primary Antibody Recognizes Antigen 1 Protein of interest is Bound to plastic Plastic Dish

29. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 4 Secondary Antibody Conjugated to an enzyme 2 Unreacted binding sites are Covered with a non-reactive protein 3 Primary Antibody Recognizes Antigen 1 Protein of interest is Bound to plastic Plastic Dish

30. Antibodies in specific analysis • ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides and other substances (drug testing) 5 Enzyme reacts with substrate producing colored product 4 Secondary Antibody Conjugated to an enzyme 2 Unreacted binding sites are Covered with a non-reactive protein 3 Primary Antibody Recognizes Antigen 1 Protein of interest is Bound to plastic Plastic Dish

31. Western blot - good for mixtures of proteins, identifying size and characteristics • transfer proteins form SDS PAGE to paper for antibody analysis. • Primary antibody recognizes protein antigen • a secondary antibody recognizes the Fc region and is conjugated to a second molecule to act as a signal