Protein Purification Lecture Notes PDF

Protein Purification Step 1. Break open cells. Step 2. Homogenize the cell lysate. Step 3. Pellet organelles and other large objects by centrifugation. Particles that reach the bottom of the tube form a pellet, while smaller particles remain in equilibrium in the supernatant. Objects with large...

Protein Purification Step 1. Break open cells. Step 2. Homogenize the cell lysate. Step 3. Pellet organelles and other large objects by centrifugation. Particles that reach the bottom of the tube form a pellet, while smaller particles remain in equilibrium in the supernatant. Objects with larger R drift faster!  Ion-Exchange Chromatography Step 4. Pass cleared lysate to a bead-packed column to separate proteins by their migration based on size, charge, affinity... Ion exchange chromatography separates proteins based on the sign and magnitude of the net electric charge. The resin uses either cation- or anion- bound charged groups. Load cleared lysate into the column. Wash the column with a low-salt buffer to remove protein not bound to the resin. Increasing the concentration of free salt ions reduces electrostatic interaction between the resin and the bound protein. Gradually increase the salt concentration gradually and start collecting the eluents from the column.  Size-Exclusion (Gel Filtration) Chromatography Separates proteins based on size. The resin contains porous beads with labyrinth-like paths inside. Molecules near the size of the pores get trapped by the beads and migrate slowly. Large molecules pass freely. Load the lysate. Do not wash (because the proteins are not bound to the column). Elute the column by flowing the buffer solution. Large proteins emerge from the column before small proteins.  Affinity Chromatography Separates based on binding affinity The protein of interest is bound to the column while other proteins are washed away. The protein is eluted by a high concentration of salt or ligand that competes with the binding of the protein to the column. Which protein would elute first from a gel filtration column? A. protein A, with Mr = 27,000 B. protein B, with Mr = 58,400 C. protein C, a homodimer with protomer Mr = 11,300 D. protein D, with Mr = 15,600 Which protein would elute first from a gel filtration column? B. protein B, with Mr = 58,400 Size-exclusion chromatography, also called gel filtration, separates proteins according to size. In this method, large proteins emerge from the column sooner than small ones do. A new protein resembling myosin was reported. Unlike myosin, it binds calcium. Its isoelectric point and molecular weight are very similar to those of myosin. Which method would BEST separate the new protein from myosin if those two proteins were in the same buffer solution? A. ion-exchange chromatography B. size-exclusion chromatography C. affinity chromatography D. dialysis E. fractionation Which method would BEST separate the new protein from myosin if those two proteins were in the same buffer solution? C. affinity chromatography Attaching calcium to the beads in the column would create an affinity matrix that could help purify the protein. Proteins that do not bind to calcium would flow more rapidly through the column than the new protein, which does bind calcium. Purity Increases by Successive Steps  Purification Steps Increase Specific Activity A Hypothetical Purification Table for an Enzyme Fraction Total Specific volume protein Activity activity Procedure or step (mL) (mg) (units) (units/mg) 1. Crude cellular extract 1,400 10,000 100,000 10 2. Precipitation with ammonium 280 3,000 96,000 32 sulfate 3. Ion-exchange chromatography 90 400 80,000 200 4. Size-exclusion 80 100 60,000 600 chromatography 5. Affinity chromatography 6 3 45,000 15,000 purification factor = final specific activity/starting specific activity = 15,000/10 = 1,500 percent yield = percentage of the final activity/starting activity = 45,000/100,000 = 45%  Gel Electrophoresis Gel electrophoresis is used to measure the molecular weight, purity, and oligomeric/complex state of proteins (and nucleic acids) Load your sample into the wells of a two-dimensional polyacrylamide gel. Apply an electric field to force the migration of the proteins into the gel. Separate the proteins by their electrophoretic mobility that depends on mass, net charge, and shape of the proteins.  Electrophoretic Mobility 𝑭𝒆𝒍𝒆𝒄 = 𝒁𝒆𝑬 𝑤ℎ𝑒𝑟𝑒 𝑍 𝑖𝑠 𝑡ℎ𝑒 𝑡𝑜𝑡𝑎𝑙 𝑛𝑒𝑡 𝑐ℎ𝑎𝑟𝑔𝑒, 𝐸 𝑖𝑠 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐 𝑓𝑖𝑒𝑙𝑑 𝑎𝑛𝑑 𝑒 𝑖𝑠 1 𝑐𝑜𝑢𝑙𝑜𝑚𝑏. 𝐹𝑑𝑟𝑖𝑓𝑡 = 𝑓𝑣𝑑𝑟𝑖𝑓𝑡 = 𝑍𝑒𝐸 𝑤ℎ𝑒𝑟𝑒 𝑓 𝑖𝑠 𝑡ℎ𝑒 𝑓𝑟𝑖𝑐𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑎𝑛𝑑 𝑣𝑑𝑟𝑖𝑓𝑡 𝑖𝑠 𝑑𝑟𝑖𝑓𝑡 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦. 𝑣𝑑𝑟𝑖𝑓𝑡 𝑍𝑒 𝒆𝒍𝒆𝒄𝒕𝒓𝒐𝒑𝒉𝒐𝒓𝒆𝒕𝒊𝒄 𝒎𝒐𝒃𝒊𝒍𝒊𝒕𝒚 𝝁 = = 𝐸 𝑓 Friction f increases by the size (R, radius) and asymmetric shape of the protein. Two different-sized molecules with the same charge-to-size ratio run with the same mobility under an electric field in water. In native gels, the proteins are not denatured. The direction and speed of mobility depend on both the size (f) and net charge (Z). Issues: Proteins vary greatly in their charge-to-mass ratio. Proteins with a zero net charge do not migrate into the gel. Proteins with a net positive charge migrate in the opposite direction. Proteins vary greatly in their shape, altering their friction.  Denatured Gels In denatured gels, proteins are denatured by heat and detergent SDS. SDS is bound per two amino acids, creating a net negative charge that correlates with the length (and hence the mass) of the peptide chain. Therefore, the mass-to-charge (m/z) becomes roughly the same for all proteins. In principle, all different-sized proteins covered with SDS would run at about the same mobility in water. However, polyacrylamide reduces the mobility of an unfolded chain linearly by log Mr of the proteins (Mr : mass). Issues: Oligomers or complexes with many proteins are split into individual peptide chains.  Blue Native Gels In blue native gels, proteins are nonspecifically coated with a negatively charged molecule (i.e Coomassie G-250) without any protein denaturation. Proteins with neutral or a net positive charge are converted to a net negative charge, allowing them to migrate into the gel. Complexes are not disassembled, and their abundance, mass, and purity can be determined.  Absorption Spectroscopy: Beer Lambert Law 𝐼 A T 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑠𝑠𝑖𝑜𝑛: 𝑇 = 𝐼0 0 100% 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑑𝑒𝑐𝑟𝑒𝑎𝑠𝑒𝑠 𝑒𝑥𝑝𝑜𝑛𝑒𝑛𝑡𝑖𝑎𝑙𝑙𝑦 𝑏𝑦 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ: 𝑇 = 𝑒 −𝜀𝑐𝑙 1 10% 2 1% 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒: 𝐴 = −𝑙𝑜𝑔𝑇 = − log 𝑒 −𝜀𝑐𝑙 = 𝜀𝑐𝑙 3 0.1% 𝑰𝟎 4 0.01% 𝑨 = 𝒍𝒐𝒈 = 𝜺𝒄𝒍 𝑰 5 0.001% 𝜀: 𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑀 −1 𝑐𝑚−1 𝑐: 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑀 The specific absorbance of your protein (or 𝑙: 𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ (𝑐𝑚) nucleic acid) is measured by subtracting the absorbance of your blank buffer!  Measuring Protein Concentration from Absorbance Major contributions to protein absorption stem from aromatic tryptophan (W) and tyrosine (Y) residues with high extinction coefficients at 280 nm. Phenylalanine (F) absorbs maximally at 260 nm but little at 280 nm. The extinction coefficient at 280 nm can be estimated using the following formula: 𝜺𝟐𝟖𝟎 = 𝒏𝑾 𝒙 𝟓, 𝟓𝟎𝟎 + 𝒏𝒀 𝒙 𝟏, 𝟒𝟗𝟎 ni is the number of corresponding residues present in the protein.  Mass Spectrometry Step 1: Ionize the sample. In electrospray ionization, molecules are passed through a needle, dispersing the solution into a fine mist of charged microdroplets. The needle is kept at a high voltage, causing positively charged ions (i.e. , H + or Na+) from the solution to be added to droplets. Step 2: The solvent surrounding the macromolecules rapidly evaporates in a vacuum, leaving multiply charged macromolecular ions in the gas phase.  Time of Flight Mass Spectrometry 𝑭𝒆𝒍𝒆𝒄 = 𝒛𝒆𝑬 𝑭𝒆𝒍𝒆𝒄 = 𝒎𝒂 𝑧𝑒𝐸 = 𝑚𝑎 𝑧 ∶ 𝑛𝑒𝑡 𝑐ℎ𝑎𝑟𝑔𝑒 𝑚: 𝑚𝑎𝑠𝑠 𝒛 𝐸: 𝑒𝑙𝑒𝑐𝑡𝑟𝑖𝑐 𝑓𝑖𝑒𝑙𝑑 𝑎: 𝑎𝑐𝑐𝑒𝑙𝑒𝑟𝑎𝑡𝑖𝑜𝑛. 𝒂 = 𝑒𝐸 𝒎 𝑒: 1 𝑐𝑜𝑢𝑙𝑜𝑚𝑏 Step 3: Separate the ions by their m/z. In time of flight, ions are accelerated linearly through an electric field in the gas phase. Ions with a lower mass to charge ratio (m/z) will be accelerated more. Deduce m/z from the time of flight of the ion before it hits the detector.  Orbitrap Ions are deflected vertically by a magnetic field due to differences in their masses. Ions with lower m/z deflect more from their path. Electron trajectory (i.e where the ion hits the detector) is converted to m/z.  Mass Spectrum of a Whole Protein Mass spectrum of a whole protein (no protease cleavage) ionized with multiple ions using electrospray ionization. All peaks have the same mass but different charges. Adjacent peaks differ by +1 charge. The mass of the protein is 47,342 Da.  Mass Spectrum of a Cleaved Protein Protease Cleavage points Trypsin Lys, Arg (C) Chymotrypsin Phe, Trp, Tyr (C) V8 protease Asp, Glu (C) Asp-N-protease Asp, Glu (N) Pepsin Leu, Phe, Trp, Tyr (N) Endoproteinase Lys (C) Cyanogen bromide Met (C) Before loading into the mass spec, the protein was cleaved into peptide fragments by trypsin. The sample was ionized with matrix-assisted laser desorption/ionization (MALDI). Most of the positive ions formed will carry a charge of z = +1. All major peaks have the same charge but differ by mass. Measures molecular mass with 1 Da accuracy!  Peptide Sequencing: Tandem MS MS-1 sorts peptides produced by proteolytic cleavage and only one of the peptides produced by cleavage emerges. The collision chamber further fragments the peptide into two pieces (b type and y type) by breaking a peptide bond. MS-2 measures m/z ratios of charged fragments. Each peak in the MS-2 spectrum has one less amino acid than the peak before. Mass differences between successive peaks reveal the peptide sequence. Which statement is true about mass spectrometry? A. Mass spectrometry can be performed on analytes in the liquid phase. B. Mass spectrometry can obtain the sequences of multiple polypeptide segments of 100 residues each. C. The mass (m) of an analyte is used to deduce the mass-to-charge ratio, m/z, with high precision. D. Mass spectrometry can monitor changes in the cellular proteome as a function of metabolic state. Which statement is true about mass spectrometry? D. Mass spectrometry can monitor changes in the cellular proteome as a function of metabolic state. When coupled to peptide separation protocols, mass spectrometry can document a complete cellular proteome. Changes in the cellular proteome can be monitored as a function of metabolic state or environmental conditions.  Determining Protein Structure: Cryo-Electron Microscopy The sample is flash-frozen in vitreous (noncrystalline) thin ice. A high-intensity electron beam is focused onto the sample. The transmitted beam is focused onto a direct electron-detector camera. 2D images of ~100,000 individual molecules are reconstructed to obtain the 3D structure at a few Å resolution!  Bioinformatics: Sequence Alignment Amino acid sequence can inform 3D structure, function, cellular location, and evolution Proteins with similar functions (homologs) have regions with well-conserved sequences. Homologs in the same species are called paralogs. Homologs in different species are called orthologs.  Bioinformatics: Sequence Alignment Sequence alignment tools assign a score based on two proteins to have identical segments (positive score) or gaps (negative score). The consensus sequence reflects the most common amino acids at each position among homologous proteins. Overall height reflects sequence conservation, and the height of individual symbols reflects the frequency of that amino acid. Amino acids with similar characteristics (charge, polar, nonpolar) are often interchangeable.