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Protein Purification (from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course). PowerPoint PPT Presentation

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Protein Purification (from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course). Object: to separate a particular protein from all other proteins and cell components. There are many proteins (over 4300 genes in E. coli )

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Protein Purification (from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course).

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Protein Purification

(from a lecture by Dr. Richard Burgess, University of Wisconsin, Madison, at the CSH protein purification course).

Object: to separate a particular protein from all other proteins and cell components

There are many proteins (over 4300 genes in E. coli)

A given protein can be 0.001-20% of total protein

Other components:

nucleic acids, carbohydrates, lipids, small molecules

Enzymes are found in different states and locations:

soluble, insoluble, membrane bound, DNA bound,

in organelles, cytoplasmic, periplasmic, nuclear

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  • Study Question

  • You are given a shoe box full of an assortment of small objects including:

    • Ping Pong balls

    • Sugar cubes

    • Paper clips

    • 1/2” brass screws

    • Iron filings

  • 1. List the properties of each of these components that might help you fractionate them.

  • 2. Devise the most efficient method you can for getting pure paper clips.

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    20 Naturally-occurring Amino Acids


    D, E, (C, Y)


    K, R, H


    I, L, V, W, F


    S, T, N, Q


    G, A, M, P

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    Overview of Protein Purification

    Types of Separations

    Protein Properties

    Protein Inactivation/Stabilization

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    Protein Analysis and Purification

    Analytical Separations




    Preparative Separations

    Various chromatographic methods

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    Total E. coli Proteins - 2-Dimensional Gel

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    Main Types of Molecular InteractionsHydrogen BondsN H - - - - NN-H + Nlow temperaturehigh temperatureN H - - - - O C strength is very dependent on geometry donor acceptor and distance (2.6-3.1 A)Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) high salt high temperaturelow salt Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+)low ionic strength high ionic strength

















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    Variables that Affect Molecular Forces


    Ionic strength

    Ion type

    Polarity of solvent (dielectric constant)


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    Protein Properties - Handles for Fractionation

    Size(110 Da/amino acid residue)

    smallest most proteinslargest

    Amino acids: 30 100 1,000 15,000

    MW (kDa): 3.3 11 110 1,600

    Multi-subunit complexes can contain 5-30 subunits


    globular (sphere) asymmetric (cigar)

    Effects frictional properties, effective radius, movement through pores


    Gel filtration

    Elutes earlier

    Appears larger

    Sediments slower

    Appears smaller

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    Protein Properties - Handles for Fractionation

    Net charge

    Ionizable grouppKapH2 pH7 pH12

    C-terminal (COOH)4.0oooooooo----------------------------------------

    Aspartate (COOH)4.5oooooooooo-------------------------------------

    Glutamate (COOH)4.6ooooooooooo------------------------------------

    Histidine (imidazole)6.2+++++++++++++oooooooooooooooooooo

    N-terminal (amino)7.3+++++++++++++++oooooooooooooooooo

    Cysteine (SH)9.3ooooooooooooooooooooooo-----------------

    Tyrosine (phenol)10.1oooooooooooooooooooooooooo-------------

    Lysine (amino)10.4++++++++++++++++++++++++oooooooo

    Arginine (guanido)12.0++++++++++++++++++++++++++++++o

    Isoelectric point

    pI = pH where protein has zero net charge

    Typical range of pI = 4-9

    Charge distribution


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    Protein Properties-Handles for Fractionation

    hydrophobic patch


    Hydrophobic residues usually are buried internally

    The number and distribution on the surface vary

    Can use Hydrophobic Interaction Chromatography


    Varies from barely soluble (<mg/ml) to very soluble (>300 mg/ml)

    Varies with pH, ionic strength/type, polarity of solvent, temperature

    Least soluble at isoelectric point where there is least charge repulsion

    Ligand and metal binding

    Affinity for cofactors, substrates, effector molecules, metals, DNA

    When ligand is immobilized on a bead, you have an affinity bead

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    Separation Processes that can be Used to Fractionate Proteins

    Separation ProcessBasis of Separation

    Precipitation ammonium sulfate solubility

    polyethyleneimine (PEI) charge, size

    isoelectric solubility, pI

    Chromatographygel filtration (SEC) size, shape

    ion exchange (IEX) charge, charge distribution

    hydrophobic interaction(HIC) hydrophobicity

    DNA affinity DNA binding site

    immunoaffinity (IAC) specific epitope

    chromatofocusing pI

    Electrophoresisgel electrophoresis (PAGE) charge, size, shape

    isoelectric focusing (IEF) pI

    Centrifugationsucrose gradient size shape, density

    Ultrafiltrationultrafiltration (UF) size, shape

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    Typical Protein Purification Scheme

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    Protein Inactivation/StabilizationBuffers Solution Components

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    Protein Sources for Purification

    Traditional natural sourcesBacteria, animal and plant tissue

    Cloning recombinant proteins into overexpression vector/host systems for intracellular production (E. coli the most used)

    In vitro protein synthesis Transcription/translation systems

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    Total E. coli Proteins - 2-Dimensional Gel

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    Determining the protein sequence from gel


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    What You Can Learn from Amino Acid Sequence1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Extinction coefficient 4. Hydrophobicity & membrane spanning regions 5. Potential modification sites 6. Conserved motifs that suggest cofactor affinityWhat You Can’t Learn from Amino Acid Sequence 1. Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge distribution, antigenic sites)Conclusion: Protein Purification is still very empirical!

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    Engineering Proteins for Ease of Purification and Detection

    Once you have a gene cloned and can over-express the protein, you can alter protein to improve the ease of purification or detection

    You can fuse a tag to the N-or C- terminus of your protein

    You can decide to remove the tag or not

    Basic strategies

    Add signal sequence that causes secretion into culture medium

    Add protein that helps the protein refold and stay soluble

    Add sequence that aids in precipitation

    Add an affinity handle (by far the most used is the His-tag)

    Add sequence that aids in detection

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    CSH Protein Course -Sigma32 Purification

    MW A B C D E F GA/3 B/3 D/3




    10 kDa



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