Protein Purification Techniques PDF

 Extracting Pure Proteins from Cells 0 Purification techniques focus mainly on size & charge 0 The first step is homogenization (grinding, Potter–Elvejhem homogenizer, sonication, freezing and thawing, detergents) 0 Differential centrifugation (600 g: unbroken cells & nuclei; 15,000 g:...

 Extracting Pure Proteins from Cells 0 Purification techniques focus mainly on size & charge 0 The first step is homogenization (grinding, Potter–Elvejhem homogenizer, sonication, freezing and thawing, detergents) 0 Differential centrifugation (600 g: unbroken cells & nuclei; 15,000 g: mitochondria; 100,000 g: ribosomes and membrane fragments  Salting in & out 0 Are proteins soluble? If yes, to which limit? 0 Salt stabilizes the various charged groups on a protein molecule and enhance the polarity of water and increases the ionic strength, thus attracting protein into the solution and enhancing the solubility of protein 0 Ammonium sulfate is the most common reagent to use at this step 0 This technique is important but results are crude  Dialysis 0 Principle of diffusion 0 Concept of MW cut-off 0 Pure vs. crude  Column Chromatography 0 Greek chroma, “color,” and graphein, “to write” 0 Is it just for colourful proteins? 0 Chromatography is based on two phases: stationary & mobile 0 Washing or Elution? 0 What are the different kinds?  Size-exclusion chromatography Gel-filtration chromatography 0 Separation on the basis of size (MW) 0 Stationary (cross-linked gel particles): consist of one of two kinds of polymers; the 1st is a carb. polymer (ex. dextran or agarose); often referred to by Sephadex and Sepharose. The 2nd is based on polyacrylamide (Bio-Gel) 0 Extent of crosslinking & pore size (exclusion limit) 0 Convenient & MW estimate 0 Each gel has range of sizes that separate linearly with the log of the molecular weight Molecular-sieve chromatography  Ion-exchange chromatography 0 Interaction based on net charge & is less specific 0 Resin is either negatively charged (cation exchanger) or positively charged (anion exchanger) 0 Buffer equilibration, exchange resin is bound to counter-ions. A cation-exchange resin is usually bound to Na+ or K+ ions, and an anion exchanger is usually bound to Cl– ions 0 Proteins mixture loading 0 Elution (higher salt concentration)  Problem 0 You have 5 different proteins (#1, #2, #3, #4, and #5), with different isoelectric points (pIs). 0 pI#5 = 2.3 0 pI#4 = 4.7 0 pI#1 = 7.2 0 pI#2 = 9.1 0 pI#3 = 12.1 0 Starting the column at pH 6.5, the sample is added and then washed to remove unbound molecules. What is the order of protein elution in a 0 Cationic-exchange chromatography? 0 An anionic exchange chromatography?  Affinity chromatography 0 It has specific binding properties 0 The polymer (stationary) is covalently linked to a ligand that binds specifically to the desired protein 0 The bound protein can be eluted by adding high conc. of the soluble ligand 0 Protein–ligand interaction can also be disrupted with a change in pH or ionic strength 0 Convenient & products are very pure (Antigen-antibody, His-tag, GST-Tag)  Electrophoresis 0 Based on the motion of charged particles in an electric field 0 Macromolecules have differing mobilities based on their charge, shape, and size 0 The most common medium is a polymer of agarose or acrylamide  Agarose or PAGE? 0 Agarose (nucleic acids), PAGE (proteins) 0 In PAGE: SDS or NO-SDS, detergent, CH3(CH2)10CH2OSO3Na+ 0 SDS completely denatures proteins (multi-subunit proteins) 0 Acrylamide offers higher resistance to large molecules 0 Shape and charge are approximately the same (sizes is the determining factor) 0 Acrylamide without the SDS (native gel): study proteins in their native conformation (mobility is not an indication of size)  Isoelectric focusing 0 Proteins have different isoelectric points 0 Gel prepared with a pH gradient parallel to electric-field gradient 0 Two-dimensional gel electrophoresis (2-D gels)  Questions 0 Describe the protein’s structure based on the following results of SDS-PAGE: 1. Under non-reducing condition, a protein exists as one 40-KDa band. Under reducing conditions, the protein exists as two 20-KDa bands. 2. Under non- reducing condition, a protein exists as two bands, 30 KDa and 20 KDa. Under reducing conditions, the protein also exists as two bands, 15 KDa and 10 KDa. 3. Under non- reducing condition, a protein exists as two bands, 40 KDa and 20 KDa. Under reducing conditions, the protein exists as one bands of 20 KDa. Under non-reducing condition, a protein exists as one 40-KDa band. Under reducing conditions, the protein exists as two 20-KDa bands. Non-reducing Reducing 40 KDa 20 KDa Under Under non- reducing condition, a protein exists as two bands, 30 KDa and 20 KDa. Under reducing conditions, the protein also exists as two bands, 15 KDa and 10 Kda. Non-reducing Reducing 30 KDa 20 KDa 15 KDa 10 KDa Under Under non- reducing condition, a protein exists as two bands, 30 KDa and 20 KDa. Under reducing conditions, the protein also exists as two bands, 15 KDa and 10 Kda. Non-reducing Reducing 100 KDa 50 KDa 25 KDa Under non- reducing condition, a protein exists as two bands, 40 KDa and 20 KDa. Under reducing conditions, the protein exists as one bands of 20 KDa. Non-reducing Reducing 40 KDa 20 KDa 20 KDa  Immunoassays - ELISA 0 Enzyme-Linked Immunosorbent Assay 0 Detect & quantify substances (peptides, proteins, antibodies & hormones) 0 Usually done in 96-well plates 0 Rapid, convenient, and sensitive (10-9 g) 0 Apllication: (Green, (No color, positive) negative) 0 Screening (HIV, Hepatitis B&C) 0 Detecting food allergens, such as milk, peanuts, walnuts, almonds, and eggs 0 Hormones (HCG, LH, TSH, T3, T4)  Protein sequencing 0 Protein sequencing is basically the process of knowing the amino sequence of a protein o a peptide. 0 One technique is known as Edman Degradation. 0 This procedure involves a step-by-step cleavage of the N- terminal residue of a peptide, allowing for the identification of each cleaved residue.  Protein sequencing - Edman Method 0 how much and which amino acids are involved 0 Hydrolysis (heating + HCl) & Separation (ion-exchange chromatography or by high performance liquid chromatography, HPLC)  Procedure 0 This method utilizes phenylisothiocyanate (PITC) to react with the N-terminal residue. 0 The resultant amino acid is hydrolyzed, liberated from the peptide, and identified by chromatographic procedures.  Advantage 0 Since the remainder of the peptide is intact, the entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide. 0 The Edman degradation technique does not allow peptides more than 50 residues to be sequenced.  Cleavage methods 0 It is possible to sequence whole proteins by cleaving them into smaller peptides. 0 This is facilitated by three methods: 0 Chemical digestion 0 Endopeptidases 0 Exopeptidases  Chemical digestion 0 The most commonly utilized chemical reagent that cleaves peptide bonds by recognition of specific amino acid residues is cyanogen bromide (CNBr). 0 This reagent causes specific cleavage at the C-terminal side of methionine residues. 0 A protein that has 10 methionine residues will usually yield 11 peptides on cleavage with CNBr.  Endopeptidases 0 These are enzymes that cleave at specific sites within the primary sequence of proteins. 0 The resultant smaller peptides can be chromatographically separated and subjected to Edman degradation sequencing reactions.  Example 0 Trypsin cleaves polypeptide chains on the carboxyl side of arginine and lysine residues. 0 A protein that contains 9 lysine and 7 arginine residues will usually yield 17 peptides on digestion with trypsin. Another example  Other examples Enzyme Specificity peptide bond C-terminal to R, K, but not Trypsin if next to P peptide bond C-terminal to F, Y, W but Chymotrypsin not if next to P peptide bond C-terminal to A, G, S, V, Elastase but not if next to P peptide bond N-terminal to L, F, W, Y, Pepsin but not when next to P  Exopeptidases 0 These are enzymes that cleave amino acids starting at the end of the peptide. 0 There are two types: 0 Aminopeptidases that cleave at the N-terminus 0 Carboxypeptidases that cleave at the C-terminus  Protein sequencing – prediction from DNA & RNA 0 If the sequence of the gene is known, this is very easy 0 If the sequence of the gene is unknown (newly isolated proteins)? Sequence a short segment, complementary RNA, isolate mRNA, PCR, gene sequencing  Determination of 3°Structure 0 X-ray crystallography 0 uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation 0 exposure to a beam of x-rays gives a series diffraction patterns 0 information on molecular coordinates is extracted by a mathematical analysis called a Fourier series 0 2-D Nuclear magnetic resonance 0 can be done on protein samples in aqueous solution X-Ray and NMR Data High resolution method to determine 3˚ Determines solution structure structure of proteins (from crystal) Structural info. Gained from Diffraction pattern produced by determining distances between nuclei electrons scattering X-rays that aid in structure determination Series of patterns taken at different Results are independent of X-ray angles gives structural information crystallography