Biochemistry
Lab
Cell Harvest Extraction and Buffer Prep Lecture Notes Handout
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Cell lysis – critical steps
Extraction of protein from cells includes specific and critical steps – a few conditions must first be considered
Is your protein easily denatured? – temperature may be a factor, include thiols for disulfide stability
Is your protein hydrophobic? – will need to include detergents throughout isolation – know which detergents to
use to avoid inhibition of your protein’s function
Is your protein a target for proteases? If so, careful inclusion of specific protease inhibitors are needed
What is your next (downstream) step? Watch the ionic concentration or inclusion of various co-factors
• NaCl – for ion exchange EDTA for His tag purification
Buffer systems
Main Components
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Buffer – find a suitable buffer in the pH range for purification and stability of your protein 10-50 mM in strength
• Tris, Mops, HEPES are most commonly used
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Salts – NaCL 25-200 mM, used to decrease weak ionic interactions between proteins and maintain ionic strength of
buffer
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Detergents – Triton X-100 or Deoxycholate – helps solubilize cell membrane and maintain hydrophobic protein
structure
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ü Chelators – EDTA (divalent cations – Fe …) EGTA (Calcium ion) 1 mM – reduces oxidation from free metals
• EDTA will interfere with His-tag binding to Nickel column
ü Reducing Agents – Dithiolthreitol (DTT) or bmercaptoethanol (bME)
• At low concentration 1 – 10 mM act to maintain disulfide bridges
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ü Other– Mg , ATP, substrates – help to maintain protein structure
ü DNA – Lysis will often denature histones resulting in an unraveling of genomic DNA – this results in a very viscous
solution –
• mechanical sheering (needle sheering, sonication) or chemical treatment (DNAseI – 1.0 µg/ml) will be
needed
ü Imidazole – may be used at low concentrations to prevent weak binding proteins loading onto Ni column
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Buffer systems
Proteases are released as soon as the cell is lysed and remain throughout most of the purification
Four main classes
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Serine proteases (trypsin, chymotrypsin & elastase)
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Cysteine proteases (papain, calpain and lysosomal cathespsins)
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Aspartic proteases (pepsin and rennin)
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Metallo-proteases thermolysisin & carboxypeptidaseA
Each class has a different set of inhibitors
Different organisms have different subsets of proteases. Thus it is important to have the right inhibitor(s) for your
expression host
Many are soluble in DMSO or alcohol and have a short half life
Lysis methods
Osmotic shock – 2 volumes of low osmotic buffer (low salt) drives water into cell and bursts periplasmic region – low
protein but low protease
Enzymatic degradation – addition of lysozyme to cleave protein-sugar coating of cell wall, often combined with
another method (freeze thaw or mechanical sheering)
Freeze thaw – Quick freezing (LN2) and repeated thaw bursts cells as ice crystals forming in cells break open
membrane – used with shock or grinding
Abrasive Grinding – blend cell paste with sand or small glass beads – mechanically disrupts cells. Good for difficult
to lyse cells and plant tissue
Sonication – used for moderate volumes of cells in suspension (5 – 50 ml). Cells are lysed by cavitation - quick
changes in pressure due to sound waves under tip.
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Causes cells to swell and shrink and eventually burst
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Temperature is a problem here. Keep on ice
French Press (valve-type processor) – Forces cell suspension through narrow needle valve under pressure
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Lyses cells by sheering
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Expensive set up but efficient for mid to large (not process) scale batches
Biochemistry
Lab
Cell Harvest Extraction and Buffer Prep Lecture Notes Handout
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Lysis Method
Cell Bomb – Place cells under high pressure using nitrogen gas
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At high pressure, nitrogen gas becomes liquid and freely diffuses into cell.
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Upon quick release of pressure, liquid inside of cell becomes gas and bursts cells
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Expensive but effective for small to mid sized cultures
One step shopping – prepared solutions
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Several companies have simple solutions which combine several components to lyse cells with minimal
work.
• Bugbuster, B-PER, CellLytic, Qproteome, Bactozol
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These can work very well, reduce need for sonication or other mechanical disruption and speed along lysis.
BUT for large scale cost can be significant