Chapter 5: Protein Isolation - Lecture Notes

Chapter 5: Protein Isolation Protein isolation requires understanding the basic physical and chemical properties of the protein. Solubility, acidity, basicity, interactions with different purification methods, detergents, salts, etc. Isolation of any protein requires a strategy! LOTS...

Chapter 5: Protein Isolation Protein isolation requires understanding the basic physical and chemical properties of the protein. Solubility, acidity, basicity, interactions with different purification methods, detergents, salts, etc. Isolation of any protein requires a strategy! LOTS OF INFORMATION IN THIS SECTION!!!  Considerations for Protein Isolation Must have sensitive method for detection. Functional assays, antibodies, analytical platforms Select a good source for the protein. a)Rich source of material. b)Baker’s yeast (Saccharomyces cerevisiae) c)E. coli Molecular cloning techniques: Over-express proteins (bacteria or mammalian cells) Purification strategy Interrelationships of purification steps to isolate the protein in the desired state (functional or not). Recombinant Proteins May Be Found in Inclusion Bodies Molecular cloning allows any protein coding genes to be introduced into selected microorganisms for expression at a very high level. Cloned protein can reach up to 40% of the total protein of the microorganism.  Stability: Proteins Can Denature!!! How can we stabilize the protein through purification steps! Denaturation is the process by which a protein loses its “native” or active shape or conformation. H-bonds, ionic interactions, van der Waals interactions, and hydrophobic interactions can be disrupted. Temperature can play a role “cold labile” “heat labile” Protein can be air-denatured - egg white meringue - absorption to surfaces (glass, plastic) Damaged by oxidation 02 Heavy metals damage proteins -they bind to protein- Cu +, Hg+ Protect against-Proteases, Inhibitors, Changes in pH Protein Purification Procedures  Purification Strategy: Protein Solubility Protein solubility depends on the concentration of dissolved ions: Ionic strength Salting in – At low ionic strength, increases in the concentration of dissolved ions leads to an increase in solubility by weakening the interaction between individual protein molecules. Interactions between protein molecules leads to aggregation (i.e. insolubility of proteins. - + + + + - - - - - + + + + - - - - + - + Salting out + + – As the ionic strength increases, they out compete the proteins for water molecules and the proteins become less soluble, aggregate, and fall out of solution.  Purification Strategy: Salting Out Use (NH4)2SO4 : it is a Very Soluble salt that does not harm proteins.  Salting Out a). At low ionic strength, all of the proteins are soluble b). As the ionic strength increases, the least soluble protein precipitates c). At even higher ionic strengths, further proteins precipitate. This process is continued until the desired protein is precipitated. Proteins are least soluble when they are This process not only allows neutral, so these salting out experiments are you to obtain the desired usually carried out at the pI of the protein of protein, it removes many interest (i.e. the isoelectric point where unwanted proteins in the pH=pI, and the net charge on the protein is 0). process Proteins are Least Soluble at Their Isoeletric Point  Quantification of a purification protocol for a fictitious protein Step Total protein Total activity Specific activity, Yield (%) Purification level (mg) (units) (units mg-1 Homogenization 15,000 150,000 10 100 1 Salt fractionation 4,600 138,000 30 92 3 Ion-exchange 1,278 115,500 90 77 9 chromatography Molecular 68.8 75,000 1,100 50 110 exclusion chromatography Affinity 1.75 52,500 30,000 35 3,000 chromatography http://www.ncbi.nlm.nih.gov/books/NBK22410/  Chromatography Analytical methods used to separate molecules. Involves a mobile and a stationary phase. Mobile phase is what the material to be separated is dissolved in. Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing binding/ interactions each molecule has with both the mobile and stationary phase. Interactions are different depending on the specific method. Chromatography  Types of Chromatography If separation is based on hydrophilic interactions, Hydrophilic Interaction (HILIC) or normal phase chromatography. If separation is based on hydrophobic interactions, reversed-phase chromatography. If separation is based on ionic interaction, Ion Exchange Chromatography. If the separation is based on size of molecule, Gel Filtration or Size Exclusion Chromatography. If the separation is based on ligand affinity, Affinity Chromatography.  Ion Exchange Chromatography A solid matrix with a positive charge, i.e., R+ can bind different anions with different affinities. We can swap one counter ion for another (R+A-) + B-  (R+B-) + A- R = Resin and exchanges Anions (-) This is an anion exchange resin - the stationary phase is decorated with positively charged groups which bind anions There are also cation exchange resins - the stationary phase is decorated with negatively charged groups which bind cations The type of an R group can determine the strength of interaction between the matrix, R and the counter ion. If R is R- (R-A+) + B+  (R-B+) + A+ Ion Exchange Chromatography  Proteins Are Charged The charge is positive below pI (more H+) The charge is negative above pI (less H+) The choice of exchange resin depends on the charge of the protein and the pH at which you want to do the purification. Once the protein binds, all unbound proteins are washed off the column. Bound proteins are eluted by increasing the ionic strength, changing the counter ion or changing the pH altering the charge on the protein or the column.  Ion Exchange Chromatography (a) The mixture of proteins applied to column (purple disc). (b) The salt concentration is low at the beginning, elute proteins with the lowest affinity for the column first (red protein) (c) The salt concentration is then increased, eluting proteins that interact more strongly with the ion exchange column. The most frequently used anion exchanger is: diethylaminoethyl (DEAE) Matrix-CH2-CH2-NH(CH2CH3)2+ The most frequently used cation exchanger is: carboxymethyl Matrix-CH2-COO-,  Hydrophobic Interaction Chromatography Reversed Phase Chromatography The chromatography column is coated with hydrophobic molecules (butyl (C4), octyl (C8), octadecyl (C18), or phenyl groups) which interact with hydrophobic residues on the surfaces of the proteins. Elution is typically increasing hydrophobic content of an aqueous solvent (increasing acetonitrile or methanol in water). Decreasing ionic strength (i.e. lower salt concentration), increasing detergent concentration (which disrupt hydrophobic interactions and lead to unfolding of the proteins), and changes in pH can also affect elution. Gel Filtration Chromatography Gel Filtration Chromatography Each gel bead consists of a gel matrix (wavy lines in the brown spheres) Small molecules (red dots) can fit into the internal spaces in the beads and get stuck Larger molecules (blue dots) cannot fit into the internal spaces in the beads and they come through the column faster  Affinity Chromatography Ligands (yellow in the figure to the left) are attached to the solid resin matrix Only one protein will have the binding site for the ligand attached to the solid resin matrix, thus highly specific! The proteins that do not have the proper ligand binding site will flow through the column fastest The desired protein (i.e. the one with the proper ligand binding site) is then recovered from the column by washing with a solution with high ligand concentration, altered ionic strength, or altered pH Example: Protein A (from Staphylococcus aureus) column to capture IgG; substrate column to capture enzyme; Ni+ column to capture His- tagged protein His-tag Affinity Chromatography Engineer a protein of interest to have a His6-tag Use Ni-NTA/Ni-TED to affinity capture the protein Use Imidazole to elute the protein Cleave the His6 tag away if desired http://www.affymetrix.com/  Quantification of a purification protocol for a fictitious protein Step Total protein Total activity Specific activity, Yield (%) Purification level (mg) (units) (units mg-1 Homogenization 15,000 150,000 10 100 1 Salt fractionation 4,600 138,000 30 92 3 Ion-exchange 1,278 115,500 90 77 9 chromatography Molecular 68.8 75,000 1,100 50 110 exclusion chromatography Affinity 1.75 52,500 30,000 35 3,000 chromatography http://www.ncbi.nlm.nih.gov/books/NBK22410/ SDS-PAGE of Proteins at different points of purification Electrophoresis (PAGE) Electrophoresis is a method for separating proteins based on how they move in an electric field Image to the left is an electrophoretogram of serum, stained with amido black The sample starts at the top, an electric field is applied, and proteins migrate The molecules at the bottom are the lightest Molecules of similar charge and size move through the gel as a band The pH is typically 9 in these experiments so most proteins have a net negative charge and move toward the positive electrode (i.e. the one attached to the bottom of the gel) Gels are typically made of polyacrylamide and so the experiment is called polyacrylamide gel electrophoresis (PAGE) SDS-PAGE Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), SDS-PAGE is used to separate protein mixtures in a protein denaturing environment (SDS – soap) That is, the SDS causes proteins to denature and take on a rodlike shape and have similar charge to mass ratios Therefore, proteins are separated by molecular mass Again, the lighter proteins travel further In the figure, several (8) protein mixtures are run at the same time, some are controls and the others are samples Each sample is in a separate column, called a “lane” SDS-PAGE  Isoelectric Focusing Proteins pI = 5.2 pI = 6.8 pI = 8.7 pH = 4 pH = 10 Immobilized pH gradient in a sheet or tube gel or recent liquid pH gradient format. Different pH ranges are available. Proteins are applied either at one end or in wells on top of the pH gradient. Proteins have a net charge at most pH values except their pI value. Applied electric field forces proteins to migrate in the direction opposite of the electric field (i.e. Positive proteins will migrate toward the negative anode). Proteins migrate through the pH gradient until they reach pH = pI, at which point they have no net charge and do not migrate further.  2D Gel Electrophoresis Combination of isoelectric focusing and SDS- PAGE First isoelectric focusing is carried out in one direction Then subjected to SDS-PAGE in the perpendicular direction  2D Gel Electrophoresis Combination of isoelectric focusing and SDS-PAGE First isoelectric focusing is carried out in one direction Then subjected to SDS-PAGE in the perpendicular direction pH4 pH6 pH8 pH10 130kd 70kd 30kd 16kd Aromatic Amino Acids are responsible for UV light (200-400 nm) absorbance Why do we use 260/280 ratio to determine DNA/RNA purity? Enzyme-linked immunosorbent assay (ELISA) When proteins are purified, there must be some way of detecting (assaying) for detecting the presence and amount of protein The assay must be performed at each purification step in order to follow protein purification Direct enzyme assay if target is an enzyme Coupled enzymatic assay if protein/enzyme cannot be detected directly Immunoassays are among the most sensitive – Use antibodies raised against the target protein (antigen) – A protein mixture containing the protein of interest can be separated since ONLY the antigenic protein will bind to the antibody – ELISA is a specialized version of an immunoassay – The binding of antigen to antibody is detected by the binding of a second antibody to the antigen that has a coupled enzymatic activity that can be detected (e.g., fluorescence, etc.)  Basics of Detection Western Blot – Detection by antibody to target protein – Dependent on availability and quality of antibodies ELISA – Enzymatic or antibody-based detection – Amenable to antibody-based approaches – Dependent on availability and quality of antibodies Mass Spectrometry – Direct coupling with antibody-based approaches – No antibodies are necessary, but helpful – Capable of ID for unexpected proteins Do chapter end problems in Chapter 5 Q3, 5, 7-10, 12, 18, 22-25 You will find a lot of similar problems in Midterm 2 Additional problems for practice 1. The following reagents are often used in protein chemistry: CNBr, Trypsin, Performic acid, Mercaptoethanol, 6N HCl, Chymotrypsin, Phenyl isothiocyanate Please match them to the tasks listed below a. Determination of the amino acid sequence of a small peptide b. Separate peptide chains of a multi-chain protein c. Hydrolysis of peptide bonds on the carboxyl side of aromatic residues d. Cleavage of peptide bonds on the carboxyl side of methionines e. Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine residues 2. The oligopeptide AVGWRVKS was digested with the enzyme trypsin. Which method would be most appropriate for separating the products, ion-exchange or gel filtration chromatography? Please explain. Suppose that the peptide was digested with chymotrypsin. What would then be the better separation approach? Please explain. 3. A mixture of four proteins need to be separated. Protein A, isoelectric point (pI)=4.1, Mass=80 kD Protein B, pI=9.0, Mass=81 kD Protein C, pI=8.8, Mass=37 kD Protein D, pI=3.9, Mass=172 kD How do you separate protein B from the mixture. If Protein B also carries a His-tag, how will you separate Protein B from the mixture?