1. BICH 605 October 6, 8, 20 & 22 Larry Dangott Department of Biochemistry and Biophysics Room 440 BioBio 845-2965 ljdangott@tamu.edu BICH 605; Fall 2009

2. BICH 605 Planning:  Method Development; Strategies  Activity Tracking; Fraction ‘pooling’ Techniques:  Electrophoresis (SDS, Isoelectric Focusing)  Chromatography (GFC, IEX, Affinity, rpHPLC)  Structural Characterization (Amino Acid Analysis; Protein Sequencing)  Proteomics (Protein ID and characterization using mass spectrometry) To present an OVERVIEW of techniques used in Protein Purification and Analysis. OUTLINE

3. It helps to know something about your protein 1.Source (organism; tissue; organelle; amount) 2.Assemblage vs. monomer 3.Cytosolic vs. membrane-bound 4.Size 5.Isoelectric point (pI) 6.Post-translational modification 7.Relative abundance Protein Purification

4. Source (organism; tissue; organelle; amount) 1.Natural vs. Recombinant (tagged?) 2.Tissue (bone (hard), blood (liquid), heart (soft), brain (fatty)); extraction 3.Organelle (nucleus, mitochondria, ER, plasma membrane); pre-fractionation 4.Amount (a LOT or a little ; scale); cost and practicality (myoglobin = easy; EGFR = hard) Protein Purification

5. Multimer vs. Monomer Affects buffer choices (assembly vs. disassembly) Affects choice of separation media (size) Cytosol vs. Membrane Affects pre-fractionation choices (extraction) Separation methods (centrifuge, columns) Affects buffer choices (detergent) Size (sort of related to Multimer vs. Monomer) Affects choice of separation media (GFC) Affects solubility (larger proteins like to precipitate) Isoelectric point (pI) Affects choice of separation media (charge) Affects solubility (precipitate at pI) Affects buffer choices (precipitation point; charge) Post-Translational Modification Affects choice of separation media (affinity) Protein Purification

6. Important Steps You May Use: Extraction (French press, sonication, detergent, homogenization) Centrifugation (low speed, ultra- speeds, differential gradient) Protein estimation method (colorimetric, spectroscopy) Protein concentrating method (salt or organic precipitation, lyophilization, membrane filtration) Chromatography (IEX, gel filtration, chromatofocusing) Electrophoresis (IEF, preparative native or SDS) Protein Purification

7. Sample Preparation Extraction (grinding, detergent lysis, sonication) Salt exchange (gel filtration, filters, dialysis) Capture Ion Exchange Affinity Hydrophobic Interaction Intermediate Purification Ion exchange Hydrophobic Interaction Polishing 1.Gel Filtration 2.Reversed phase Protein Purification A COMPLEX STRATEGY FOR PROTEIN PURIFICATION A SIMPLE STRATEGY His-tag: affinity

8. Systematic method development requires..... Defining a way of quantifying, or at least identifying, the presence of your target molecule, and of assessing its purity. Don’t rely solely on literature (or coworker) statements. Verify yourself. 50% success rate. Keep a record of your purification process. Notebook, notebook, notebook……….... Protein Purification Happy Boss

9. Our Example: Enzyme Purification There are two major objectives in enzyme purification: To obtain the highest SPECIFIC ACTIVITY possible, measured as activity per unit protein To obtain the MAXIMUM YIELD of enzymatic activity. (Theoretically, this is 100%. Practically, one is usually happy to settle for something like 30%.) Protein Purification

10. When purifying a protein, one wants to keep track of how one is doing relative to the two major objectives. Therefore, at each step, one must measure: 1.Volume 2.Protein concentration (colorimetric assay, UV) 3.Enzyme activity (units/ml; specific to ‘your’ protein) Protein Purification

11. These measurements are combined in the calculation of: Total activity = Enzyme activity/aliquot volume X Total volume Total protein = Protein/aliquot volume X Total volume Specific activity = Enzyme activity in an aliquot/Amt of Protein in the aliquot (THIS IS THE BIG ONE) (In measurements of total activity and protein, remember to adjust for volumes set aside for various reasons. If this is not done, the yield will be artificially low).

12. Vol X Activity Units/vol = Total Activity Units Vol X mg/ml = Total Protein (mg) Calculate Activity Units and Total Protein Use to calculate Specific Activity Divide Total Activity Units by Total Protein (mg) = Specific Activity in Units/protein (mg)

13. Divide current Specific Activity by Initial Specific Activity = Fold Purified Divide current Total Activity Units by Original Activity Units = % Yield Fold purification goes UP Yield goes DOWN KNOWING WHICH FRACTIONS TO POOL IS IMPORTANT

14. Selecting Fractions based on Specific Activity and SDS PAGE Mutant Tyrosine Hydroxylase; Ion Exchange; NaCl Gradient Pure Mutant TyrOH has a V max of ~12 PoolRNA?Stable? Data courtesy of Colette Daubner; Fitzpatrick Lab

15. The less prevalent the protein is in the cytosol, the higher the degree of purification that will be required for its purification to homogeneity. For example: A protein that is 50% of the cellular protein needs to be purified only 2-fold. In contrast: A protein that is only 0.1% of the cellular protein needs to be purified 1000-fold. Protein Purification

16. 1.Sample Preparation 1.Extraction (grinding, detergent lysis, sonication) 2.Salt exchange (gel filtration, filters, dialysis) 2.Capture 1.Ion Exchange 2.Affinity 3.Hydrophobic Interaction 3.Intermediate Purification 1.Ion exchange 2.Hydrophobic Interaction 4.Polishing 1.Gel Filtration 2.Reversed phase Mode of monitoring the purification……………… Protein Purification A TYPICAL STRATEGY FOR PROTEIN PURIFICATION

17. Tracking your protein is critically important. How do you know where it is? Biological Assay (usually specific; extremely sensitive; slower) Binding Assay (usually specific; sensitive, semi-automate) Chemical Assay (colorimetric assays, enzyme assays) Physical Assay (mass spec, UV spectrometry) Protein Purification Separation Assay (electrophoresis)

18. Electrophoresis is a electrically driven sieving process used to separate complex mixtures of proteins. Can be ANALYTICAL or PREPARATIVE. SDS PAGE is used to investigate subunit composition and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further micro- analytical applications Principle of SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) Most proteins bind the ionic detergent, SDS (sodium dodecyl sulfate), in a constant weight-to-detergent ratio, leading to identical negative charge density per mass for the denatured proteins and a uniform shape. Thus, theoretically, SDS-protein complexes migrate through a solid matrix (polyacrylamide) and are separated according to size, not charge. SDS PAGE ELECTROPHORETIC ANALYSIS OF PROTEINS

19. APPLICATIONS Polypeptide composition and fraction profiling: Purified protein complexes or multimeric proteins consisting of subunits of different molecular size will be resolved into constituent polypeptides. Screen fractions during protein purification. Quaternary structure profile: Comparison of the protein bands obtained under non-reducing and reducing conditions provides information about the molecular size of subunits and protein complexes. Size estimation: The relationship between the relative mobility and log molecular weight is linear over some range. With the use of plots like those shown here, the molecular weight of an unknown protein (or its' subunits) may be determined by comparison with known protein standards. SDS PAGE

20. In SDS gel electrophoresis, negatively charged, SDS-coated proteins migrate in response to an electrical field through pores in a crosslinked polyacrylamide gel matrix Pore size decreases with higher acrylamide concentrations Smaller pores are used for smaller proteins/peptides; larger pore sizes are used for larger proteins.

21. PROCEDURE Proteins to be analyzed are solubilized and denatured by boiling (or heating) in the presence of SDS and reducing reagent, an aliquot of the protein solution is applied to a gel lane, and the individual proteins are separated electrophoretically. The reducing reagent β- Mercaptoethanol (  -ME) or (dithiothreitol (DTT)) is added during solubilization to reduce disulfide bonds. SDS PAGE

22. The polyacrylamide gel is cast as a separating gel (sometimes called the resolving or running gel) topped by a stacking gel and secured in an electrophoresis apparatus (see figure). The stacking gel is run at slightly acid pH (6.8). The separating gel is run at pH 8.8. The stacking gel is ~4% acrylamide and the separating gel is a higher concentration. The stacker brings the proteins to a common ‘starting line’ and the separator sieves them apart. The concentration of acrylamide in the separating gel is determined by the range of molecular weights of interest. SDS PAGE

23. Tris-Glycine in Upper Buffer Tris-HCl pH 6.8 in Stacking Gel Tris-HCl pH 8.8 in Separating Gel SDS PAGE

24. Glycine equilibria SDS PAGE

25. Formation of an ion front SDS PAGE

26. It is the voltage gradient that sharpens the ion boundary SDS PAGE

27. What happens to proteins? SDS PAGE

28. In separating gel Glycine mobility increases, becomes greater than protein mobility, but still slower than Cl - SDS PAGE

29. Protein sample, now in a narrow band, encounters both the increase in pH and decrease in pore size. Increase in pH would tend to increase electrophoretic mobility, but smaller pores decrease mobility. Relative rate of movement of ions in separating gel is chloride > glycinate > protein. Proteins separate based on charge/mass ratio and on size and shape parameters. SDS PAGE

30. PROTEIN DETECTION Detection limitFixing? Coomassie Blue G-250 or R-250 staining50 ngfixing Silver1 ngfixing Fluorescent stains(Sypro)10 ngnon-fixing Negative stains (zinc, copper)1- 10 ngnon-fixing SDS PAGE Coomassie Blue Silver Sypro Ruby

31. Relative Mobility (Rf) Molecular Weight (Log Scale) SIZE ESTIMATION SDS PAGE IMPORTANT MW ESTIMATION BY SDS-PAGE IS ONLY APPROXIMATE AND IS REFERRED TO AS APPARENT MOLECULAR WEIGHT. Unusual protein compositions or physical properties can cause anomalous mobilities during SDS-PAGE. SDS gels can be used as a micro- purification step and the individual polypeptides can be isolated from the gel by electroelution or electroblotting and the amino acid sequences can be determined or peptide maps obtained.

32. ISOELECTRIC FOCUSING Isoelectric Point (pI) is specific pH at which net charge equals zero At pI, protein has no net charge and will not migrate in an electric field IEF is a technique to separate proteins based on Isoelectric Point (native or denatured)

33. Isoelectric Focusing IEF CAN BE PERFORMED WITH MOBILE pH GRADIENTS OR IMMOBILIZED pH GRADIENTS Mobile gradients are prepared with Carrier Ampholytes (CAs) (mixed polymers (300-1000 Da in size) mixed with solid support (mobile). Immobile gradients are prepared by covalently coupling Ampholytes to solid support and blending. Solid support is usually polyacrylamide but can be agarose for preparative purposes

34. IMMOBILIZED pH GRADIENT IEFMOBILE pH GRADIENT IEF Isoelectric Focusing ADVANTAGES of IMMOBILIZED GRADIENTS Stable pH gradients Ease of handling Reproducibility Extreme pH resolution

35. Combine IEF & SDS PAGE High Resolution Zoom gels (pH range) Detect isoforms Post-translational modifications Expression Proteomics 2 Dimensional Gel Electrophoresis

36. Combine IEF & SDS PAGE High Resolution Zoom gels (pH range) ANALYTICAL Detect isoforms Post-translational modifications PREPARATIVE Mass spectrometry 2 Dimensional Gel Electrophoresis

37. END OF DAY 1