Purificacion Proteinas.pdf

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  • 7/29/2019 Purificacion Proteinas.pdf


    Protein Purification

    Principles and Methods

  • 7/29/2019 Purificacion Proteinas.pdf



    Complex, polymeric, asymmetric and sensitive molecules

    Contain covalent bound prosthetic groups and non-covalentbound cofactors

    Many non-covalent bounds e.g. Hydrogen-Bounds, Dipol-

    Interactions and Hydrophobic-Interactions

    Weak interactions are important for structure and function

    (activity) of the protein

    In most cases the purification must be gentle!

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    Before the purification

    Cultivation of bacteria

    Cell disruption: Periplasmic and cytoplasmic proteins are released

    Centrifugation leads to a soluble fraction (supernatant) which containsall soluble periplasmic and cytoplasmic proteins and a membranefraction from which membrane bound proteins can be solubilised withdetergents (e.g. Triton X-100)

    The soluble or membrane fraction are the start point of the furtherpurification by chromatography

    Cell disruption: French Press



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    French Press

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    Membrane Proteins

    Peripheral membrane proteins: in most cases soluble in buffers withhigh or low ionic strength or high pH

    Integral membrane proteins: they contain trans membrane helices andmust be solubilised to conserve conformation and function of theprotein

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    Solubilisation of integral membrane proteins

    Solubilisation of proteins is done with a detergents concentration abovethe CMC to ensure the incorporation of membrane lipid into detergentmicelles.

    CMC = critical micelle concentration

    depends on temperature, ionic strength and pH of the buffer andconcentration of uncharged substances like urea or alcohol

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    Some detergents

    Ionic detergents:

    Sodium-Dodecylsulfate: denatures Proteins ( SDS-PAGE)

    Na-Deoxycholate: preticipates by pH

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    Which proteins are purified?

    Metabolic pathways

    Energy production

    Aim: biochemical characterisation (Reactivity, subunit composition,organic and inorganic cofactors, 3D structure)

    and why?

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    Purification strategies

    Protein stabilisation:

    Integral membrane Proteins: Solubilisation

    Purification at 4C: reduces protease activity

    Addition of protease inhibitors: commonly used are EDTA andPMSF (toxic)

    Quickly load on first column after cell disruption and ultracentrifuge

    Main impurities are removed first, lesser in the second or third step

    Max 5% impurities are acceptable

    In general: Max 4 purification steps

    No steps with purification factor < 5

    No steps with < 30% yield

    No steps which last longer than one day and one night

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    Separation material in columns, streamed by buffer

    liquid chromatography

    HPLC: high pressure/performance liquid chromatographyFPLC: fast protein liquid chromatography

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    FPLC unit

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    Separation principles


    size exclusion chromatography (= gel filtration, = gel permeationchromatography)


    anion or cation exchange chromatography; chromatofocusing

    Hydrophobicityhydrophobic interaction chromatography (HIC)


    affinity chromatography


    ammonium sulfate precipitation (non chromatographic, rel. imprecise)

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    Size exclusion chromatography

    Different pore sizes, depending on the size of the proteins

    Separation is based in diffusion slow flow rate

    Pressure sensitive materials

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    Example for a separation by gel filtration

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    Noteworthy about chromatography

    Gel filtration:

    limited sample volume: 2-3 mllimited flow rate gel filtraion takes timedelution of the sample by a factor of about 3

    low purificatopn factor: 3-6needs column buffer with high ionic strength: min 0.1 M

    Ion exchage chromatography:

    To remember: Protein binding depents on electrostatic interactions withthe column material.Strength of binding depents on pH and ionic strength ofthe buffer, the pI of the protein and the charge density onthe column.

    In general: Technical easier than gel filtration: columns could bepacked at the FPLC.

    Sample volume can be multiple times the column volume.Higher flow rates.Purification factor: 3-15

    Sample gets concentrated. Dont use charged detergents!

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    Ion exchage chromatography

    Anion exchanger Cation exchanger

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    Binding behaviour of proteins

    pI = isoelectric point of the protein the pH value at which the possesno net charge

    The pI determines the charge of the protein at a given pH

    pH > pI negative net charge

    pH < pI positive net charge

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    pI and protein separation on ion exchange columns

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    pI is not alone responsible for the binding behaviour of proteins

    Binding is sometimes influenced by local charges and not by the net

    charge of the protein

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    Example for a separation by ion exchange chromatography

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    Carrier materials

    Poly sugars

    Sepharose (Agarose), Sephadex (Dextran), Sephacel (Cellulose)

    Rare sugars can hardly be utilised by bacteria.

    Low flow rates Material changes its Volume depending on the ionic strangth

    Material settles over time

    Beads Polystyrol/Divinylbenzen beads with charge carrier added

    Relative high flow rates

    Good pressure stability, no compression

    Hard charges could stress the protein

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    Beads and tentacle

    Charges are linked with flexible spacers to polymeric beads. This leads to asoft binding of proteins despite of hard charges on the matrix

    Relative high flow rates Good pressure stability

    High capaticity

    Perfusion beads Porous material, beads with chanels

    Very big surface

    Highly pressure stable

    Very high flow rates

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