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Introduction to protein purification IGP methodology 2005-2006

. Why does the protein of interest need to be purified?What is the source of the protein?What is known about the protein? . Important questions to ask before you embark. High resolution structure or therapeutic use?>99% purity is requiredAntibody generation?High yield of 90-95% pure proteinBiochemical assays?Degree of yield/purity varies with application.

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Introduction to protein purification IGP methodology 2005-2006

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    1. Introduction to protein purification IGP methodology 2005-2006 Pranav Danthi PostDoc/Dermody lab pranav.danthi@vanderbilt.edu

    2. Important questions to ask before you embark

    3. High resolution structure or therapeutic use? >99% purity is required Antibody generation? High yield of 90-95% pure protein Biochemical assays? Degree of yield/purity varies with application Why are proteins purified?

    4. Sources of protein Non-engineered Examples: Organ tissues or Cell lines The sequence of the protein may be unknown and purification is based on the activity of the protein Abundance of target protein is low Engineered Examples: Expressed in Bacteria, Yeast, Insect or mammalian cell lines (transfected or transduced) The gene sequence for the protein is known and the gene for the protein is cloned Relatively higher abundance of protein Over-expressed proteins can accumulate in inclusion bodies

    6. Quantification of purification Yield How much protein of interest is in the fraction? Amount of protein of interest Purity (Specific activity) What fraction of purified protein is the protein of interest? Protein of interest/total protein

    7. Measuring proteins Protein of Interest Activity assay - Enzyme assay - Bioassay Western blot (quantitative) - Need specific antibodies and standard curve Total protein Absorbance at 280nm - non-destructive Colorimetric assays Color is directly proportional to protein concentration Reaction is destructive Reaction is sensitive to buffer conditions

    8. Quantification of purification

    10. Purification method Batch method - good for small scale purification quick and dirty Gradient elution not possible (would require stepwise change) Column Chromatography Large scale purification possible Columns give better resolution Can be automated using pump or FPLC

    11. Typical setup of FPLC

    12. FPLC instrumentation

    13. Protein purification is a multi-step process

    15. Types of purification Take advantage of biophysical characteristics Selective Precipitation/ Capture (based on solubility/ specific affinity) Capture/Intermediate Gradient Chromatography (based on charge properties) Capture/Intermediate/Polishing Size-exclusion Chromatography (based on size or shape) Intermediate/Polishing

    17. Salting out of proteins using ammonium sulfate

    18. Selective capture using affinity chromatography Enzyme: Substrate, inhibitor, cofactor Antibody: Antigen, virus, cell Lectin: glycoprotein, cell receptor Nucleic Acids, Heparin: histone, polymerases Hormone, vitamin: Receptor, carriers

    19. Affinity Chromatography

    20. Affinity chromatography of recombinant proteins

    21. Example of a tagging vector

    22. Chromatogram from Ni affinity purification

    23. Protein over-expression may lead to aggregate formation A large amount of over-expressed protein is found in the insoluble fraction Can modify conditions of induction or growth to reduce inclusion body formation Can isolate protein from insoluble fraction using denaturing conditions Proteins may then be refolded if desired after purification

    26. Summary Before you start Set the aims (purity and quantity) Characterize the target protein Develop assay methods Protocol development checklist Select techniques and conditions compatible with sample stability. Use combinations of different separation principles. Start with high selectivity – increase efficiency. Use few steps. Limit sample handling between purification step

    27. Problems You are purifying a his-tagged protein from E. Coli. lysate (lane L). The material from two different purifications are shown. Which (sample A or B) is purer? Why do you think so? If the material eluted looked like C what changes would you make to your affinity purification conditions to make it as pure as A or B?

    28. Is a purification technique that gives you a 60% yield but only a 3-fold purification better than one that gives you a 10-fold purification but only a 25% yield? You are purifying a protein and you consistently see a band that reacts with an antibody specific to your protein of interest but is of much smaller size, what may the possible reasons for this?

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