Protein purification


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Protein purification

  1. 1. Protein Purification tutorial 3rd week 2nd year biochemists christiane riedinger
  2. 2. Somerville College 2nd year Biochemistry Michaelmas 2008 Protein Purification 1. Reading material Your assignment: Please take a look at some of the following resources. There is no need to read them all, but of course it’s not discouraged… - Voet & Voet, “Biochemistry”, chapter 5 - R.K. Scopes, “Protein Purification: Principles and Practice” - Allan Jones, “Practical Skills in the Biomolecular Sciences” - Notes from Prof Endicott’s lectures in Michaelmas term (see WebLearn for past lectures) - Search for reviews in Methods in Enzymology - The internet! ;-) 2. Techniques in protein purification Can each of you please pick one topic from the ones below: Affinity Chromatography Ion Exchange Chromatography Size Exclusion Chromatography Methods to determine protein concentration Methods to assess protein purity Please prepare an overview about the topic of your choice. It should contain the most important aspects, but should be no longer than one A4 page. During the tutorial, you will present this to your fellow students, so please bring along 6 copies of it. 3. Develop your own purification strategy Please prepare a practical protocol for the purification of the protein SLP-X. Indicate the main steps of purification Reading list: in a flow chart and describe each sub-step in more detail, explaining the method used and why you chose it. Ideally, you would like to have ~10mg of pure (>95%) enzymatically active (!) protein at the end of the process. what did you find most helpful? An enzyme, SLP-X, found in sheep liver has an interesting dehydrogenase activity that you would like to investigate further. Your collaborators have done some initial characterization of SLP-X and found the following: (Please note: not all of this information is necessarily useful!) • SLP-X is a cytoplasmic protein expressed at ~2mg/kg in sheep liver and it has not been detected in any other tissues tested. A local butcher has offered you a large amount of sheep liver. • Your collaborators have raised a monoclonal antibody (IgG) directed against SLP-X. They have kindly offered you 3 mg of the antibody. • Your collaborators have worked out a simple and robust protocol for testing SLP-X enzymatic activity. • On an SDS gel (and confirmed using Western-blot analysis) SLP-X runs as a single band of molecular weight of approximately 30 kD • SLP-X has a pI of 5.2. • SLP-X has no disulfide bonds or cysteine residues. • SLP-X has a single glycosylation site with high mannose content. • SLP-X absorbs strongly at 280 nm. • The enzyme activity of SLP-X is lost at high temperatures. Initial tests have suggested that the protein is stable for months at 4oC but only minutes at 37oC. Good luck!
  3. 3. Overview 1. general considerations / strategy 2. purification techniques - chromatography methods - other methods 3. protein detection 4. assessment of purity
  4. 4. Purpose of Protein Purification 1. - increasing stability - functional studies - structural studies - large scale production
  5. 5. Choosing a strategy: 1. ...characteristics of the protein - primary sequence: - oligomeric state - modifications (glycosylation, phosphorylation..) - interactions with co-factors, ions - cytosolic, external matrix, membrane protein? - expressed soluble/insoluble?
  6. 6. Choosing a strategy: 1. ...cloning / choosing the right vector - express tagged/untagged? - allow for affinity chromatography - possibly improve solubility - potential “tags” to consider: - GST - poly-HIS - strep - HA - maltose binding protein - ...
  7. 7. Useful tools for primary sequence analysis 1.
  8. 8. 1. submit sequence result molecular weight: small/large? extinction (important for SEC, coefficient: SDS PAGE) good absorbance at 280nm? pI: cation or anion exchange? stability: needs protease sequence: inhibitors? temperature? hydrophobic/hydrophilic? solubility? acidic/alkaline?
  9. 9. 1. Protein purification is a multi-step procedure: Based on protein properties choose number and type of individual purification steps separation technique, i.e. affinity chromatography ion exchange chromatography original sample size exclusion chromatography is protein pure? reverse phase chromatography is protein active? i.e. from cell extract, tissue, blood, peptide synthesis... NO YES - concentrate - use purify further After each step assess protein purity and activity.
  10. 10. 1. Purifying insoluble proteins: - some proteins cannot be expressed solubly, even after optimisation of expression procedure and vector - does not have to be a disadvantage! - over-expressed insoluble proteins form cellular aggregates = inclusion bodies - not much purification needed - but refolding can be a problem - potential procedure: - over-expression of proteins - cell disruption - centrifugation: this time keep pellet, discard supernatant! repeated washing of pellet & centrifugation (with buffer containing detergent to remove hydrophobic cell parts such as membranes) isolated inclusion bodies: solubilise protein with high concentration of denaturing agent (urea, guanidine hydrochloride) centrifugation (discard pellet) further purification (affinity tags such as HIS-tags that work under denaturing conditions are a common first step) refold protein!! potential further polishing step
  11. 11. 2. Principles of Chromatography Throw it in the Rhine! This is what the professor of analytical chemistry during my undergrad used to say. It is a strange analogy, but it does work.. kind of! Throw a mixture of rocks that you’d like to separate into the rhine. Depending on the speed with which the rhine flows (the mobile phase) and the rockiness of its bed (the stationary phase), your different sample components will travel different distances. Let’s say you throw your sample into the Rhine at the Bodensee, component A will travel till Karlsruhe, B until Wiesbaden, and C will make it all the way to the Nothern Sea!!!
  12. 12. 2. Column Chromatography Solid Phase / Stationary Phase Mobile Phase Can be a gas (GC), a liquid (LPLC, FPLC, HPLC).
  13. 13. 2. Main Chromatography Methods Affinity Chromatography Separation by specific binding Ion Exchange Chromatography Separation by charge Size Exclusion Chromatography Separation by size/shape Reverse Phase Chromatography Separation by hydrophobicity ...batch vs column/continuous flow
  14. 14. 2. Common Features of Chromatography column protein contaminants 1. load 2. bind/exchange (wash) 3. elute (either by retention time, or by competition)
  15. 15. 2. Affinity Chromatography - separation by specific binding of the protein of interest to the column - using an immobilised specific ligand for the protein of interest or its purification tag - this ligand is usually immobilised on an agarose bead matrix - for example His, GST, HA, GFP, ... but also specific antibody against protein - bind, wash, elute - elution through addition of compound to mobile phase that competes with protein for binding to stationary phase - this compound may have to be removed from the eluate through additional purification step -
  16. 16. 2. Ion Exchange Chromatography - separation through binding of protein to column via electrostatic interaction - Column matrix is either positive (=Anion Exchange Chromatography, to bind negative ions), or negative (=Cation Exchange Chromatography, to bind positive ions) - depending on pI of protein and buffer chose anion or cation exchange - carefully chose a buffer which does not bind to column! - protein elution through use of a gradient of increasing ionic strength to weaken binding of protein to column - Some Common Ion Exchange Matrices (and Buffers)
  17. 17. 2. Size Exclusion Chromatography - also called gel filtration - separation based on mw/size - column bed often consists of agarose beads with pores of a certain size determining the separation range - as sample flows through, small components diffuse in an out of pores, while larger components cannot enter the pores and flow right through - proteins are sorted by size/shape, big proteins are eluted before small proteins. column bed size exclusion chromatography profile sample
  18. 18. 2. Reverse Phase Chromatography - separation based on hydrophobic interaction of sample with column - often used for peptides rather than large protein, as proteins can unfold when binding to column - column bed is composed of carbon-hydrogen chains of different length, often attached via Si - samples are eluted through increasing gradient of hydrophobic solvent (i.e. acetonitrile) to compete sample off the column - often followed by lyophilisation step to remove solvent
  19. 19. 2. Other Purification Methods - Salt Precipitation: By adding ammonium sulfate. Masks ionic interactions between proteins, proteins precipitate due to hydrophobic interactions. Good for enzymes. - Hydrophobic Precipitation: Using solvents. Reduce the solubility of proteins by favouring protein-protein rather than protein-solvent interactions. Danger of denaturation. - Dialysis: Exchanges every particle below a molecular weight cut-off by osmosis. Good for buffer exchange or refolding. - Lyophilisation: Removes solvent to leave only solid particles. Typically used for peptides in organic solvents. - Centrifugation: Separate proteins by molecular weight.
  20. 20. Methods to determine the protein concentration 3. - Biuret essay: Peptide bonds react with alkaline copper sulfate to form a purple compound which absorbs at 540nm. Sensitivity 1mg. - Lowry assay: Purple compound from Biuret assay reacts with Folin-Ciocalteu reagent (phosphomolybdate- phosphotungstate) to form a blue compound which absorbs at 650nm. Sensitivity 5ug. - Bradford assay: Coomassie Brilliant blue binds to positively charged residues in protein. This shifts the absorbance wavelength of the dye from 465 to 595nm. Sensitivity 1-20ug. - Spectrophotometric assay: Uses aromatic residues in protein sequence which absorb at 280nm. (also possible: peptide bond absorbance at 214nm). Keep in mind that many other substances absorb in that area. - Amino Acid composition analysis: Destroy protein and measure amino acid content by HPLC.Very accurate method, useful to determine extinction coefficient (as opposed to prediction, which assumes random coil).
  21. 21. 4. Assess Purity of Sample - SDS PAGE Gel Electrophoresis: Mix protein sample with loading buffer containing sodium dodecyl sulphate (SDS), a positively charged solvent that binds all along the protein’s amino acid chain. A polyacrylamide gel then separates sample components by size only, as mass to charge ratio is now constant. Small proteins migrate faster than big ones. - Mass Spectrometry: SDS-PAGE gel Separate proteins by size. More in Fridays tutorial!!! - Western Blot: Assessment of activity. (Figure from PIERCE webpage) lane1: molecular weight marker
  22. 22. Your purification exercise.
  23. 23. The End. Any Suggestions/Questions?