Protein Purification

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Protein Purification

• BL4010 10.10.05

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The basic techniques

• Concentration (size)
• precipitation
• ultrafiltration
• dialysis
• centrifugation
• Chromatography (size/charge/chemistry)
• ion exchange
• size exclusion
• affinity
• hydrophobic interaction

• Electrophoresis (size/charge)
• "native"
• denaturing
• isoelectric focusing
• 2-dimensional
• Immunological
• chromatography
• in situ imaging
• immunoblotting

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Electrophoresis (SDS-PAGE)

• Tris-glycine buffer
• 10 SDS

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Electrophoresis
Prestained markers have dyes covalently bound
BEFORE electrophoresis - increased MW
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Electrophoresis

• Protein detection using dyes
• Coomassie blue
• Sypro
• Cybergreen
• Silver staining

Staining with dyes AFTER electrophoresis - no
change in MW non-covalent interaction
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Western blotting

• Separate proteins by electrophoresis
• Transfer to membrane (e.g. nitrocellulose)
• Bind primary antibody
• Bind secondary antibody
• Detection

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Immuno-Affinity Chromatography

• antibody fixed to matrix
• protein binds to antibody
• wash unbound and loosely bound proteins off
  column
• elute protein with change in salt/pH

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Hydrophobic interaction chromatography

• Hydrophobic group bound to solid phase
• Binding
• high salt (increases water surface tension,
  decreases available water molecules, increases
  hydrophobic interactions)
• Elution
• decrease salt
• add detergent
• decrease polarity
• of mobile phase

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Assay and Specific Activity
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Criteria for purity

• When is protein pure or pure enough?
• homogeneity
• protein complexes?
• constant specific activity
• Practical further attempts at purification are
  futile since the only material left in the
  fraction is the material that actually is
  responsible for the activity being assayed.

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Protein purification simuation

• http//www.tlsu.leeds.ac.uk/courses/bioc2060/prote
  inlab102/proteinlab.html

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Enzymes

• BL4010 10.12.05

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Objectives

• What is an enzyme?
• How do enzymes work?
• energetics
• underlying general mechanism
• components (prosthetic groups, coenzymes)
• specific mechanisms
• Ch.13.1, 13.2, 14.1, 14.2, 14.3, 14.4, 14.5

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What is an enzyme?

• Macromolecular biological catalyst
• Can be protein or RNA

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What is an enzyme?

• Macromolecular biological catalyst
• What is a catalyst?
• is not altered by reaction
• participates but emerges unchanged
• increases the rate at which substrates and
  products reach equilibrium
• does not alter equilibrium

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Why enzymes?

• Why invest energy and resources into creating a
  large catalyst?
• Enzymes endow cells with the remarkable capacity
  to exert kinetic control over thermodynamic
  potentiality
• Fine tune selectivity (substrate binding
  specificity)
• Fine tune catalytic rate
• Additional regulatory control (e.g. allostery,
  signalling networks)

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Enzymes are good catalysts

• Enzymes can accelerate reactions as much as 1016
  over uncatalyzed rates!
• Urease is a good example
• Catalyzed rate 3x104/sec
• Uncatalyzed rate 3x10 -10/sec
• Ratio is 1x1014 !

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Enzymes are selective catalysts

• Enzymes selectively recognize proper substrates
  over other molecules
• Enzymes produce products in very high yields -
  often much greater than 95
• Specificity is controlled by structure - the
  unique fit of substrate with enzyme controls the
  selectivity for substrate and the product yield

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How do enzymes work?

• How do catalysts in general work?

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The transition state

• Understand the difference between ?G and ?G
• The overall free energy change for a reaction is
  related to the equilibrium constant
• The free energy of activation for a reaction is
  related to the rate constant
• It is extremely important to appreciate this
  distinction!

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How do enzymes work?

• Enzymes accelerate reactions by lowering the free
  energy of activation
• HOW?

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Four contributing factors to enzyme catalysisNO
ONE MECHANISM ACCOUNTS FOR CATALYSIS ALONE!

• Specific substrate binding
• local concentration of reactants
• productive orientation of reactants
• binding energy used to offset loss of entropy
• Control over solvent interactions
• desolvation (binding energy offsets)
• ordered solvent in binding pocket
• Induction of strain on reactants
• Alternate reactive pathway
• transient involvement of enzyme functional groups

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How do enzymes work?

• Enzymes accelerate reactions by lowering the free
  energy of activation
• Enzymes do this by binding the transition state
  of the reaction better than the substrate