PROTEIN PURIFICATION

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PROTEIN PURIFICATION

• Step 1Crude Extract

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E.Coli Cell Paste
Cell disruption
Pellet
Centrifugation
Crude Extract (Supernatant)
Aliquot
Ammonium Sulfate Precipitation
Centrifugation
Supernatant
Aliquot
Pellet (Resuspend)
Dialysis
Pellet
Centrifugation
Aliquot
Dialysate
Ion Exchange Chromatography
Collect fractions- assay
Assays for Specific Activity
SDS PAGE Western Blotting
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Overview OfThis Purification

• Making the Extract
• Purification Steps
• Ammonium sulfate precipitation (salting out)
• Dialysis
• Ion Exchange Chromatography

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Overview OfThis Purification Cont

• Analysis and Verification
• Assays for specific activity
• Polyacrylamide gel electrophoresis
• Western Blotting

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Making The Extract

• Homogenize the source material
• Goal get target protein into solution
• Source material
• Any biological material which contains abundant
  source of target protein
• If youre lucky
• Target protein is secreted outside the cell
• Most proteins found inside cell

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Cell Disruption

• Enzymatic
• Mechanical
• Grinding
• Homogenizing
• Motorized or not
• High pressure
• Sonication

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(No Transcript)
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Enzymatic MethodBug Buster from Novagen
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French Press
This
Not this
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Manton GaulinHomogeniser
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Sonication

• Uses high frequency sound waves to break open
  bacteria
• Generates heat
• Need ethanol ice bath
• Buffer is protective

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Avestin EmusiflexC5

• Can process 160 liters

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Two ImportantThings

• Method to assay protein
• Method to stabilize protein during the
  purification

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Proteases

• Cell lysis during cell disruption releases
  proteases from cellular compartment (lysosomes)
• How do we keep them from destroying target
  protein?
• Keep cold (4C)
• Some protocols add protease inhibitors

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Choice OfExtraction Buffer

• Buffering pH
• Based on pH stability range of protein
• Ionic strength
• Divalent cations

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Choice OfExtraction Buffer

• Reducing agents
• Protease inhibitors
• Reference and resource
• Seidman and Moore

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After Cell Disruption

• Centrifuge the homogenate to create the
• CRUDE EXTRACT
• Take aliquots
• Store at 4C
• Save the pellet (Why?)

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Components OfLaboratory Solutions

• Buffers
• Different pH optima
• Usually pH 6-8

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• Salts
• Ionic strength
• Affects 3-D structure
• Affects solubility
• Because it affects electrostatic interactions
  between side chains
• Mg, Zn, Fe

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• Co-enzymes
• NAD, etc

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Components OfLaboratory Solutions

• Divalent cations
• Ca, Mg
• Reducing agents
• Dithiothreitol, ß-mercaptoethanol
• Protease inhibitors
• Leupeptin
• PMSF
• Etc.

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To Learn More.

• Protein Analysis and Purification
• Ian M Rosenberg
• Reference and resource
• Seidman and Moore
• Many web resources