Protein Purification- Aldolase

    I. General Protein Purification Theory-
            Classical Procedure for a Novel Protein
      A.    Start with Cell/Tissue that Contains Protein (enzyme) of Interest
      1.    Why use muscle as source?
       

          -Muscles- anaerobic conditions- gycolysis occurs here

          -Glucose in blood; enters cells; broken down into pyruvate for      energy

          -muscles convert pyruvate into reduced end product (lactate) through homolactic fermentation

      2.    Where in cell is aldolase?

          a. cytosol, membrane bound, subcellular organelle?

          b. how does this affect purification?
      B.    Apply Fractionation Steps
      1.    Low Cost; High Capacity
             High Cost; Low Capacity

      2.    Types of Fractionation:
              a. Selective ppt. (i.e. (NH4)2SO4 )
              b. Chromatography (i.e. P-cell)
      C.    Assay Fractions for Protein
       

      1. Would use an enzyme assay if location of protein is unknown (i.e. something like the assay used in kinetics)

      2. Since aldolase has been purified before we know which fractions should contain the protein.

      D.    Pool Fractions with desired protein and Repeat steps B & C

E.     Determine yield and purity at each step
      -Use specific activity of enzyme to determine % yield
    II.    Specifics of Aldolase Purification
      A.    Lyse Cells

              1. Use buffer of KOH w/ EDTA

              2. How does this work?

                  -osmotic lysis

                  -soluble protin

                  -aldolase is then in solution

              3. Pellet to Remove Insoluble Material

              4. Repeat to extract more Aldolase

      B.    Ammonium Sulfate Fractionation

              1. Different Proteins have varying solubility in (NH4)2SO4

              2. Typical Protocol- Ammonium Sulfate Cuts

                  a.    Add (NH4)2SO4 to desired % saturation
                  b.    Allow proteins to ppt.
                  c.    Centrifuge to collect proteins
                  d.    The 1st ppt should be at a (NH4)2SO4 conc. where your desired
                           protein doesnít ppt.
                  e.    Increase salt conc. to a % which ppt. your desired protein
                  f.     Allow protein to ppt. and centrifuge to collect

          3. The solubility of a protein in (NH4)2SO4 often depends on protein conc.

          4. Theory of Ammonium Sulfate precipitation
              "Salting Out"

                  a.    Makes use of surface electrostatic heterogeneity

                  b.    Proteins interact with H2O in solution
                  c.    Hydrophobic patches on proteins are sticky and may lead to
                           aggregation under conditions which reduce ability of H2O to
                           solvate hydrophobic patches.
                  d.    At high salt conc. the effective conc. of H2O is decreased
                  e.    The ordered H2O molecules solvating hydrophobic patched tend
                           to be displaced, being replaced by protein-protein interactions
                           (aggregation)
                  f.    This type of aggregation is often reversible
       

    III.    General Protein Treatment
      A. Keep it Cold

      B. Donít stir vigorously or vortex
          -causes oxidation or surface denaturation

C. Donít expose proteins to extreme pH or High Temp
    -promotes denaturation D. Avoid fungal and bacterial growth if storing protein for a long time

E. Filter buffers, degas and use glass distilled H2O

F. pH of Buffer is important

         IV. Specifics of week 1 protein purification
      A.    Write out protocol step by step

      B.    For the 10í stirs use glass rods and stir by hand

      C.    Filter though glass wool "web"

      D.    Break up muscle to allow for best interaction w/ liquid

      E.    Check pH w/ meters
              -keep solution cold while doing this
              -stir (w/ stir bar & plate) while pH-ing

      F.    Donít forget to take fractions

      G.    Add Solid (NH4)2SO4 SLOWLY
              -Pour in 5-10g, let disolve, add more
              -Shouldnít have undissolved (NH4)2SO4 crystals in bottom of beaker

      H.    Think about volumes and use appropriate size beakers
              -give yourself some space

      I. KEEP THINGS COLD

      How to do this?