Unformatted text preview: Protein Puriﬁcation and Analysis
(02/15/11) General approach to purifying proteins Protein solubility Chromatography Electrophoresis Ultracentrifugation Protein Puriﬁcation
•Must have sensitive method for detection. •Select a good source for the protein. a. Rich source of material. i.e. Heart mitochondria for cytochrome C b. baker’s yeast (Saccharomyces cerevisiae) c. E. coli •Tissue speciﬁcity: Brain vs. kidney vs. eye. Chickens, cows, pigs or rats are often used. Molecular cloning techniques have allowed biochemists to over-express target proteins in bacteria by isolating the gene and placing it into a host system (over-expression strain). The ﬁrst step - get it out of the cell Methods of solubilizing cells
•Cells can be lysed by hypotonic shock. Cells with high salt inside and no salt outside will swell and rupture. •Bacteria outer membranes must be digested. - Gramnegative bacteria Hen egg white lysozyme digests (1-4) linkages in the (glycosidic bonds) of polysaccharides. •Mechanical breakage – blenders, homogenizers •French press - high pressure 20,000 lbs/in2 forced through a small hole disrupts cells •Ultrasound or sonication disrupts cells. Stability: proteins can denature!!
•H-bonds, ionic interactions, van der Waals interactions, and hydrophobic interactions can be disrupted. •Denaturation is the process by which a protein loses its “native” or active shape or conformation. •Temperature can play a role •“cold labile” •“heat labile” •Protect against-Proteases, Inhibitors, Changes in pH •Protein can be air-denatured - egg white meringue absorption to surfaces (glass, plastic) •Damaged by oxidation O2 •Heavy metals damage proteins -they bind to protein- Cu, Hg •Bacterial contamination can destroy the protein Stabilizing Proteins
pH Temperature Degradative enzymes Effect/Consideration
Proteins are isolated and kept at a pH at which they are stable/active. Proteins are often unstable at high temperatures and so are normally purified at 4°C. Must adjust pH or temperature to inactivate degradative enzymes present in the cells so that they don’t degrade the protein of interest. Add anti-proteases. Proteins are denatured by contact to air-water interfaces or by contact with glass or plastic surfaces so they are kept relatively concentrated All above conditions must be observed. Protein solutions are sometimes stored under nitrogen or argon and/or frozen at low temps. (-70 to –196 oC). Absorption to surfaces Long-term storage Assaying Proteins
•When proteins are purified, there must be some way of detecting (assaying) for 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 Enzyme-linked immunosorbent assay (ELISA) • 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.) Concentration of proteins in solution
Absorbance spectroscopy Bradford assay A = log(I/I0) = cl Strategy of Purification
Fractionation procedures or steps to isolate protein based on physical/chemical characteristics.
Characteristic Charge Polarity Procedure 1. Ion exchange, 2. Electrophoresis, 3. Isoelectric focusing 1. Adsorption chromatography 2. Paper chromatography 3. Reverse phase chromatography 4. Hydrophobic interaction 1. Dialysis and ultraﬁltration, 2. Gel electrophoresis, 3. Gel ﬁltration, 4. Ultracentrifugation 1. Afﬁnity chromatography 2. Immunopuriﬁcation 1.Salt precipitation 2. Detergent solubilization Size Speciﬁcity Solubility Ionic Strength
1 I =2 (c i Z i 2 ) Ci = the molar concentration of the ith species Zi = its ionic charge For monovalent ions, I (the ionic strength) is the same as M (the molar concentration) 1M Na+ ClZi = +1 Na+ Zi = -1 Cl- [1M * (1) 2 ]Na + [1M * ( 1) 2 ]Cl 1 + 1 I= = =1 2 2
For di- or tri-valent ions, where I is different than M. 1M MgCl2 Mg2+ = 1M, and Z = +2 while Cl- = 2M, and Z = -1 I= [1M * (2) 2 ]Mg + [2M * ( 1) 2 ]Cl 2 4+2 = =3 2 Protein Solubility
• • Since proteins contain a number of charged groups, its solubility depends on the concentration of dissolved ions 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, ions compete the proteins for water molecules and the proteins become less soluble, aggregate, and fall out of solution. Salting out
Use (NH4)2SO4 : it is a Very Soluble salt that does not harm proteins. Solubility of carboxy-hemoglobin at its isoelectric point Solubility of -lactoglobulin as a function of pH • • • • • Proteins are least soluble when they are neutral, so these salting out experiments are usually carried out at the pI of the protein (i.e. the isoelectric point where pH=pI, and the net charge on the protein is 0) 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. This process not only allows you to obtain the desired protein, it removes many unwanted proteins in the process What amino acids must predominate in Pepsin and Lysozyme to generate such pIs? 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. Types of chromatography
•Gas - liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. •Liquid - Liquid: Mobile phase is liquid, stationary phase is liquid usually bound to a solid matrix. • If separation is based on ionic interaction the method is called Ion Exchange Chromatography. •If separation is based on solubility differences between the phases the method is called Adsorption Chromatography. •If the separation is base on size of molecule the method is called Gel Filtration or Size Exclusion. •If the separation is base on ligand affinity the method is called 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+B-) + A(R+A-) + BR = Resin and exchanges Anions (-) •This is an anion exchange resin – the stationary phase is decorated with positively charged groups which bind anions in the mobile phase •There are also cation exchange resins. 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+ Proteins have a net charge
Net charge + pH The charge is positive below pI, while the charge is negative above pI _ •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
• The tan region is the ion exchange resin • The mixture of proteins is the purple disc in a). • The salt concentration is low at the beginning so the proteins with the lowest affinity for the column go through first (red protein) • The salt concentration is then increased, washing off the proteins that interact more strongly with the ion exchange medium in the column • The most frequently used anion exchanger is: diethylaminoethyl (DEAE) Matrix-CH2-CH2-NH(CH2CH3)2+ • The most frequently used cation exchanger is: carboxymethly Matrix-CH2-COO- pH? 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 Gel filtration can be used to determine the molecular mass of proteins Hydrophobic Interaction Chromatography
• The chromatography column is coated lightly with hydrophobic molecules (octyl or phenyl groups) which interact with hydrophobic residues on the surfaces of the proteins in the eluant • The eluant typically is an aqueous buffer with decreasing salt concentration (hydrophobic interactions are augmented by increases in ionic strength), increasing detergent concentration (which disrupt hydrophobic interactions and lead to unfolding of the proteins), and changes in pH Affinity Chromatography
• Ligands (yellow in the figure to the left) are attached to the solid resin matrix • The proteins in the eluant have ligand binding sites, however, only one of them will have the binding site for the ligand attached to the solid resin matrix • 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 Afﬁnity Chromatography
Based on molecular complementary between an enzyme and substrate. The substrate (R) is linked to a matrix with a spacer arm Only protein that binds R will stick to column. Put citrate on column citrate dehydrogenase will speciﬁcally bind. Add excess citrate and the enzyme will be released. Electrophoresis
• Electrophoresis is a method for separating proteins based on how they move in an electric field • 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) Polyacrylamide Gel electrophoresis (PAGE) Fig. adapted from NCBI » Bookshelf » Molecular Biology of the Cell » Introduction to the Cell » How Cells Are Studied 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” • • • • • 2D-elecrophoresis
2D electrophoresis resolves complex mixture of proteins: 1st - a sample of proteins is subjected to IEF ( isoelectric focusing) in one direction 2nd - separated proteins are subjected to SDS-PAGE in the perpendicular direction. Valuable tool for proteomics Q2: (a) In what order would the amino acids Arg, His, and Leu be eluted from a carboxymethyl (CM) column at pH6? (b) In what order would the amino acids Glu, Lys, and Val be eluted from a diethylaminoethyl (DEAE) column at pH8? Q4. Determine the subunit composition of a protein from the following information: Molecular mass by gel filtration: 200 kD Molecular mass by SDS-PAGE w/o ME: 100 kD Molecular mass by SDS-PAGE w ME: 40 kD and 60 kD ...
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This note was uploaded on 02/15/2011 for the course BCHS 3304 taught by Professor Johnson during the Spring '08 term at University of Houston.
- Spring '08