Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification

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<h1>Large Scale Protein Expression &#38; Purification</h1>
<h1>Large Scale Protein Expression &#38; Purification</h1>
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<p>Insert Text Here</p>
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<h2>Reagents and Materials</h2>
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<ul>
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<li>Autoclaved 1 L LB in 2.8 L flask </li>
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<li> Plates of protein producing bacteria being grown</li>
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<li>Shaker</li>
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<li>1 mM IPTG</li>
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<li>Appropriate antibiotics</li>
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<li>Homogenizer</li>
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<li>Shaker</li>
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<li>AKTA</li>
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<li>Proteinase Inhibitor Cocktail </li>
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<li>Shaker</li>
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<li> 10mM HEPES buffer</li>
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</ul>
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<h2>Protocol</h2>
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<ol>
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<li>Culture 1L protein-producing bacteria (strain BL21 or ER2566) in the LB with appropriate antibiotics.</li>
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<li>Grow for 12 hours at 37°C with shaking and check OD.</li>
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<li>Induce with IPTG to a final concentration of 0.1mM when OD is 0.6.</li>
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<li>Let it grow for 4 hours at 30°C.</li>
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<li>Spin down the cells for 1 hour at 8000 rpm at 4C.</li>
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<li>Resuspend cells in minimal volume lysis buffer (Lysis buffer composed of: 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0). Keep on ice.</li>
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<li>Break open cells by passing supernatant through a homogenizer 3 times at a pressure of 40psa.  Add one proteinase inhibitor cocktail per 1L of cells (COMPLETE proteinase inhibitor cocktail tablets, Roche Diagnostics). </li>
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<li>Run the purified elutions on SDS-PAGE gel.</li>
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<li>Spin homogenate for 1 hour at 18,000 rpm at 4C, then filter with minimum 0.45um pore size.</li>
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<li>Filtered crude lysate was run through the AKTA purifier through a 1ml HisTrap column at 1ml/minute in order to bind his-tagged recombinant proteins to the column. Column was washed with 10ml of lysis buffer without imiziadole. In order to elute proteins off the column, the column was washed with elution buffer gradient of 0% elution buffer up to 100% elution buffer over 20ml, then with 5ml of 100% elution buffer. Elution buffer is composed of 50mM NaH2PO4, 300mM NaCl and 250mM imizadole, pH 8.0.</li>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/1/10/JamieFigureOne.png" style="width: 70%;"></img>
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<figcaption>
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<p><b>Figure 1.</b> Screen capture of AKTA output.  Blue line corresponds with UV output. Initial high UV corresponds to flow through past column of other non-his tagged proteins in the crude lysate. Peak at 180-200 minutes corresponds to protein of interest coming off the column. Light green line corresponds to percent elution buffer running through the column. As can be seen, protein is eluted from the column at approximately 30-50% elution buffer. Fractionation coordinates are seen at the bottom of the screen in order to easily locate fractions with protein.</p>
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</figcaption>
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</figure>
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<li>Run the purified elutions on SDS-PAGE gel to assess protein size and purity.</li>
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<li>Concentrate the proteins and store the proteins in 10 mM HEPES buffer, pH 8.0</li>
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</ol>
</section>
</section>
</html>
</html>

Latest revision as of 01:39, 29 October 2013

Large Scale Protein Expression & Purification

Reagents and Materials

  • Autoclaved 1 L LB in 2.8 L flask
  • Plates of protein producing bacteria being grown
  • Shaker
  • 1 mM IPTG
  • Appropriate antibiotics
  • Homogenizer
  • Shaker
  • AKTA
  • Proteinase Inhibitor Cocktail
  • Shaker
  • 10mM HEPES buffer

Protocol

  1. Culture 1L protein-producing bacteria (strain BL21 or ER2566) in the LB with appropriate antibiotics.
  2. Grow for 12 hours at 37°C with shaking and check OD.
  3. Induce with IPTG to a final concentration of 0.1mM when OD is 0.6.
  4. Let it grow for 4 hours at 30°C.
  5. Spin down the cells for 1 hour at 8000 rpm at 4C.
  6. Resuspend cells in minimal volume lysis buffer (Lysis buffer composed of: 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0). Keep on ice.
  7. Break open cells by passing supernatant through a homogenizer 3 times at a pressure of 40psa. Add one proteinase inhibitor cocktail per 1L of cells (COMPLETE proteinase inhibitor cocktail tablets, Roche Diagnostics).
  8. Run the purified elutions on SDS-PAGE gel.
  9. Spin homogenate for 1 hour at 18,000 rpm at 4C, then filter with minimum 0.45um pore size.
  10. Filtered crude lysate was run through the AKTA purifier through a 1ml HisTrap column at 1ml/minute in order to bind his-tagged recombinant proteins to the column. Column was washed with 10ml of lysis buffer without imiziadole. In order to elute proteins off the column, the column was washed with elution buffer gradient of 0% elution buffer up to 100% elution buffer over 20ml, then with 5ml of 100% elution buffer. Elution buffer is composed of 50mM NaH2PO4, 300mM NaCl and 250mM imizadole, pH 8.0.

    11. Figure 1. Screen capture of AKTA output. Blue line corresponds with UV output. Initial high UV corresponds to flow through past column of other non-his tagged proteins in the crude lysate. Peak at 180-200 minutes corresponds to protein of interest coming off the column. Light green line corresponds to percent elution buffer running through the column. As can be seen, protein is eluted from the column at approximately 30-50% elution buffer. Fractionation coordinates are seen at the bottom of the screen in order to easily locate fractions with protein.

  11. Run the purified elutions on SDS-PAGE gel to assess protein size and purity.
  12. Concentrate the proteins and store the proteins in 10 mM HEPES buffer, pH 8.0

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