Protein Analysis and Purification: Separation Methods and Techniques

1. Protein Analysis and Purification Analytical Separations Gel-electrophoresis IEF 2D-gels Preparative Separations Various chromatographic methods

2. Total E. coli Proteins - 2-Dimensional Gel

3. Overview of Protein Purification Introduction Protein Properties Types of Separations Protein Inactivation/Stabilization Overexpression of Recombinant Proteins Purification Tags/Affinity Handles

4. Protein Purification Object: to separate a particular protein from all other proteins and cell components There are many proteins (over 4300 genes in E. coli) A given protein can be 0.001-20% of total protein Other components: nucleic acids, carbohydrates, lipids, small molecules Enzymes are found in different states and locations: soluble, insoluble, membrane bound, DNA bound, in organelles, cytoplasmic, periplasmic, nuclear

5. Scale of Purification Micro ng-mg Normal lab mg-100 mg Large lab-scale gm Pilot plant 10 g-kg Industrial 100 tons Most common in research lab

6. Amount & Purity Required for Different Uses Use Amount neededPurity Required Immunology mg-mg Polyclonal antibodies High Monoclonal antibodies Medium Enzymology mg High (>95%) Physical Properties mg-g High Enzyme/protein Chemistry mg-g High Structure g High Research Enzymes mg-mg Variable; must be free of key contaminants Pharmaceutical g-kg Very high (>99.99%) free of bacterial endotoxin Industrial Enzymes kg-ton Variable, often low

7. Study Question #1 • You are given a shoe box full of an assortment of small objects including: • Ping Pong balls • Sugar cubes • Paper clips • 1/2” brass screws • Iron filings • 1. List the properties of each of these components that might help you fractionate them. • 2. Devise the most efficient method you can for getting pure paper clips.

8. 20 Naturally-occurring Amino Acids Acidic: D, E, (C, Y) Basic: K, R, H Hydrophobic: I, L, V, W, F Polar: S, T, N, Q Other: G, A, M, P

9. Main Types of Molecular InteractionsHydrogen BondsN H - - - - NN-H + N low temperature high temperatureN H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A)Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) high salt high temperature low salt Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)low ionic strength high ionic strength H H H H H2O H H H H Cl- Na+ ... + - + -

10. Variables that Affect Molecular Forces Temperature Ionic strength Ion type Polarity of solvent (dielectric constant) pH

11. Protein Properties - Handles for Fractionation Size(110 Da/amino acid residue) smallest most proteins largest Amino acids: 30 100 1,000 15,000 MW (kDa): 3.3 11 110 1,600 Multi-subunit complexes can contain 5-30 subunits Shape globular (sphere) asymmetric (cigar) Effects frictional properties, effective radius, movement through pores Centrifuge Gel filtration Elutes earlier Appears larger Sediments slower Appears smaller

12. + - + + + + - + uniform clustered - + - - - - + - Protein Properties - Handles for Fractionation Net charge Ionizable grouppKa pH2 pH7 pH12 C-terminal (COOH) 4.0 oooooooo---------------------------------------- Aspartate (COOH) 4.5 oooooooooo------------------------------------- Glutamate (COOH) 4.6 ooooooooooo------------------------------------ Histidine (imidazole) 6.2 +++++++++++++oooooooooooooooooooo N-terminal (amino) 7.3 +++++++++++++++oooooooooooooooooo Cysteine (SH) 9.3 ooooooooooooooooooooooo----------------- Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo------------- Lysine (amino) 10.4 ++++++++++++++++++++++++oooooooo Arginine (guanido) 12.0 ++++++++++++++++++++++++++++++o Isoelectric point pI = pH where protein has zero net charge Typical range of pI = 4-9 Charge distribution versus

13. H H H Protein Properties-Handles for Fractionation hydrophobic patch Hydrophobicity Hydrophobic residues usually are buried internally The number and distribution on the surface vary Can use Hydrophobic Interaction Chromatography Solubility Varies from barely soluble (<mg/ml) to very soluble (>300 mg/ml) Varies with pH, ionic strength/type, polarity of solvent, temperature Least soluble at isoelectric point where there is least charge repulsion Ligand and metal binding Affinity for cofactors, substrates, effector molecules, metals, DNA When ligand is immobilized on a bead, you have an affinity bead

14. Protein Properties-Handles for Fractionation Monomer (0.3M NaCl) Dimer (0.05 M NaCl) Reversible association e.g., E. coli RNA polymerase Post-translational modifications Carbohydrates, lipids, phosphates, sulfates Can be very useful purification handles E.g.: Use of plant lectins to bind certain glycoproteins Specific sequence or structure Precise geometric presentation of amino acids on surface of a protein Epitope for binding to a specific antibody; use immunoaffinity column Binding site for another protein; use protein affinity column

15. Separation Processes that can be Used to Fractionate Proteins Separation Process Basis of Separation Precipitation ammonium sulfate solubility polyethyleneimine (PEI) charge, size isoelectric solubility, pI Chromatography gel filtration (SEC) size, shape ion exchange (IEX) charge, charge distribution hydrophobic interaction(HIC) hydrophobicity DNA affinity DNA binding site immunoaffinity (IAC) specific epitope chromatofocusing pI Electrophoresis gel electrophoresis (PAGE) charge, size, shape isoelectric focusing (IEF) pI Centrifugationsucrose gradient size shape, density Ultrafiltrationultrafiltration (UF) size, shape

16. Typical Protein Purification Scheme

17. Protein Inactivation/StabilizationBuffers Solution Components

18. Protein Sources for Purification Traditional natural sourcesBacteria, animal and plant tissue Cloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used) In vitro protein synthesis Transcription/translation systems

19. Total E. coli Proteins - 2-Dimensional Gel

20. What You Can Learn from Amino Acid Sequence1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinityWhat You Can’t Learn from Amino Acid Sequence 1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge distribution, antigenic sites)Conclusion: Protein Purification is still very empirical!

21. Engineering Proteins for Ease of Purification and Detection Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection You can fuse a tag to the N-or C- terminus of your protein You can decide to remove the tag or not Basic strategies Add signal sequence that causes secretion into culture medium Add protein that helps the protein refold and stay soluble Add sequence that aids in precipitation Add an affinity handle (by far the most used is the His-tag) Add sequence that aids in detection

22. CSH Protein Course -Sigma32 Purification MW A B C D E F G A/3 B/3 D/3 225 50 35 10 kDa bb’ s32