biology-class-12-unit-16-chapter-05-industrial-scale-production-of-proteins-lecture-5-6-wrbft71j2o4

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<h3 id="successpoints-to-considersuccess"><Success>Points to consider</Success></h3>
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<li>Bulk purchase of chemicals and other raw materials would bring down costs</li>
<li>The labour cost decreases sharply with increase in production</li>
<li>Material used should be inert, resistant to corrosion: stainless steel and not glass</li>
<li>Should not be leaching toxic material</li>
<li>Should follow good manufacturing practices and approvals from regulatory authority</li>
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<h3 id="successisoelectric-focussingsuccess"><Success>Isoelectric focussing</Success></h3>
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<p><strong>2D gel electrophoresis</strong></p>
<p><strong>Protein Characterization</strong></p>
<p>Includes detection of Impurities, Mass, Isoelectric charge, amino-acid sequence etc</p>
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<li><strong>Electrophoretic techniques</strong></li>
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<h3 id="successmass-spectrometrysuccess"><Success>Mass spectrometry</Success></h3>
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<ul>
<li>A mass spectrometer is an analytical device that determines the molecular weight of chemical compounds by separating molecular ions according to their mass/charge ratio (m/z) ratios</li>
<li>The molecular ions are generated either by a loss or gain of a charge (e.g. electron ejection, protonation or deprotonation)</li>
<li>After the ions are formed they can be separated according to their m/z ratio and finally detected</li>
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<h3 id="successmass-spectrometrysuccess-1"><Success>Mass spectrometry</Success></h3>
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<li>The process of ionisation, ion separation and detection in a mass spectrum can provide molecular weight or even structural information</li>
<li>A sample M with a molecular weight greater than 1200 D can give rise to multiple charged ions such as (M+nH)n+</li>
<li>Proteins/peptides have many suitable sites for protonation as all the backbone amide nitrogen atoms could be protonated theoretically as well as certain amino acid side chains such as lysine and arginine which contain primary amine functional groups</li>
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<h3 id="successmass-spectrometrysuccess-3"><Success>Mass spectrometry</Success></h3>
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<p><strong>Question-</strong> An <em>E. coli</em> cell produces at least 2000 different proteins. One of these is our enzyme of Interest produced at a level of 3000 molecules per cell under optimum conditions. If we have to purify 1g of this intra-cellular enzyme, estimate how many cells of bacteria will be required theoretically? It is given that the molecular weight of the enzyme of interest is 1,00,000</p>
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<h3 id="successmass-spectrometrysuccess-6"><Success>Mass spectrometry</Success></h3>
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<p><strong>Question.</strong> Assuming that the bacterial cell is a cylinder (d = 1µm, h=2µm) calculate</p>
<ul>
<li>
<p>The total packed cell volume of E.coli required to produce 1 g of intra-cellular enzyme</p>
</li>
<li>
<p>The volume of the fermentor required if the maximum cell concentration inside the fermentor is 5%
(cells need space to multiply)</p>
</li>
</ul>
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<h3 id="successmass-spectrometrysuccess-7"><Success>Mass spectrometry</Success></h3>
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<p><strong>Answer.</strong></p>
<p>Given d=1µm, therefore, r=0.5 µm, and H=2µm</p>
<p>Now, Volume of a (cylinder) single bacterium = πr<sup>2</sup> h = 3.142x0.5 x 10<sup>6</sup>x 0.5 x 10<sup>6</sup>x 2 x 10<sup>6</sup> (1µm=10<sup>6</sup>m) = 1.57 x 10<sup>23</sup>m³</p>
<p>From the previous example we calculated that for 1g protein 2.007 x10<sup>15</sup> cells would be required.
Now, the volume of 2.007 x 10<sup>15</sup> cells would be  —– 2.007 x10<sup>15</sup> x 1.57 x<sup>-18</sup> m³</p>
<p>=3.15 x 10<sup>-3</sup> m<sup>3</sup> = 3.15 L</p>
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<h4 id="successproteins-purification-stepsdownstream-processingsuccess"><Success>Proteins purification steps:Downstream processing</Success></h4>
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<li>The sources material chosen will dicate the range and type of contaminants present in thge starting material</li>
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<ol>
<li>If the protein in <strong>extracellur</strong>,then one needs to separate the cellular components and process the medium to isolate the protein of interest</li>
<li>However if the protein is <strong>intracellular</strong> then the choice of method of cell disruption will depend on the cell type</li>
<li>Plant and fungal cells require harsher breakage methods,animal cells are easier to break because of no cell wall.Bacterial cells being very small require high pressure techniques</li>
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<h4 id="successproteins-purification-stepsdownstream-processingsuccess-1"><Success>Proteins purification steps:Downstream processing</Success></h4>
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<p>When a crude cell homogenate is added to a biphasic mixture of dextran and polyethylene glycol (PEG) the cellular debris partitions to the lower, more polar and dense phase, dextran. Separation of the two phases achieves effective separation of cellular debris from soluble protein</p>
<ul>
<li>Effective removal of nucleic acids may be achieved by precipitation or by treatment with nucleases</li>
<li>The lipid contaminant layer can be removed by passage of solution through glasswool or cloth of very fine mesh size</li>
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<h4 id="successproteins-purification-stepsdownstream-processingsuccess-2"><Success>Proteins purification steps:Downstream processing</Success></h4>
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<p>Efforts to maintain stability of the protein during purification</p>
<ol>
<li><strong>Buffering component:</strong> It is important that during extraction proteins are provided with well buffered environment so that they can cope up with sudden changes in pH</li>
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<h4 id="successproteins-purification-stepsdownstream-processingsuccess-4"><Success>Proteins purification steps:Downstream processing</Success></h4>
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<ol start="3">
<li>
<p><strong>Inclusion of Additives in extraction:</strong> Salt, Osmolytes, reducing agents, inhibitors of proteases</p>
</li>
<li>
<p><strong>Avoidance of physical and chemical shock:</strong> agitation or addition of chemicals which may denature the target protein.</p>
</li>
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<p><strong>Minimize time</strong></p>
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