biology-class-12-unit-16-chapter-04-protein-finger-printing-peptide-mapping-lecture-4-6-6d8iqj9ha04

<h3 id="successhemoglobin-and-sickle-cell-hemoglobinsuccess-1"><Success>Hemoglobin and sickle cell hemoglobin</Success></h3>
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<li>Pure $\mathrm{Hb}$ and $\mathrm{scHb}$ are taken separately into test tubes</li>
<li>Digested with trypsin which cleaves the protein after basic amino acid residues Arg and Lys</li>
<li>Two separate strips of Whatman filter paper are spotted with $\mathrm{Hb}$ and $\mathrm{scHb}$ tryptic peptides and the peptides allowed to separate using the technique of paper electrophoresis at $\mathrm{pH} ~ 2.0$. Highly charged peptides will migrate more towards the anode/cathode</li>
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<h3 id="successhemoglobin-and-sickle-cell-hemoglobinsuccess-3"><Success>Hemoglobin and sickle cell hemoglobin</Success></h3>
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<li>They are dried and stained with a suitable visualisation reagent like Ninhydrin wherein peptide containing regions appear as orange yellow spots</li>
<li>The peptide map for $\mathrm{Hb}$ and $\mathrm{scHb}$ are compared and it was found that one peptide was differently placed in the $\mathrm{scHb}$ map</li>
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<h3 id="successhemoglobin-and-sickle-cell-hemoglobinsuccess-4"><Success>Hemoglobin and sickle cell hemoglobin</Success></h3>
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<li>On examining this peptide and determining its amino acid sequence, Ingram found that it had a valine substitution for glutamic acid in the peptide</li>
<li>The single substitution of valine for glutamic acid (val/glu are at the 6th position of the haemoglobin beta chain) dramatically changes the structure of $\mathrm{scHb}$ making it form fibres within the RBC resulting in the deformation of the cell (sickling)</li>
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<h3 id="successpeptide-mapping-o-farrelsuccess"><Success>Peptide mapping (O’ Farrel)</Success></h3>
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<ul>
<li>Peptide mapping became a useful technique to compare similar proteins from different sources</li>
<li>Slowly the information became too vast and computers were used to store this data into databases so that homology searches could be made</li>
<li>The protein fingerprinting data has been further augmented with new databases containing 2-D electrophoresis patterns of entire proteins from a given cell type, a technique developed by O’ Farrel</li>
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<h3 id="successpurification-of-proteinssuccess"><Success>Purification of proteins</Success></h3>
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<p>Isolation of a protein from a microbial culture, plant and animal sources involves various separation techniques These steps are collectively known as <strong>downstream processing</strong></p>
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<p>The exact details of the purification scheme for any given protein will depend upon a number of factors such as</p>
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<p>Exact source material chosen and location of the target protein (intracellular or extracellular)</p>
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<p>Quantity of protein required and hence amount of raw material processed</p>
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<p>Physical, chemical and biological properties of the protein</p>
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<h3 id="successsources-of-proteinssuccess"><Success>Sources of Proteins</Success></h3>
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<p>In spite of a large biodiversity of microbes we are restricted to certain bacteria/organisms which can be used as a source of protein as well as for introducing genes</p>
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<p>These microorganisms are designated as “generally regarded as safe” (GRAS). GRAS listed organisms are non-pathogenic, non-toxic and generally should not produce any antibiotics</p>
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<p>Similarily, plant tissue derived enzymes which have application in the food industry must be obtained from only non-toxic, edible plant species</p>
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<h3 id="successsources-of-proteinssuccess-1"><Success>Sources of Proteins</Success></h3>
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<p>One of the best known industrially useful enzymes is papain obtained from the latex of the green fruit and leaves of the Papaya tree</p>
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<p>This enzyme finds application in meat tenderisation, clarification of beverages, digestive aids and wound cleaning solutions</p>
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<h3 id="successrecombinant-proteinssuccess"><Success>Recombinant proteins</Success></h3>
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<p>Insulin- a peptide hormone obtained from the pancreas of cows and pigs till the 1980’s</p>
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<p>The estimated number of diabetic patients in India in the year 2020 was 20 million</p>
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<p>Fortunately, the advent of genetic engineering has ensured the availability of recombinant human insulin expressed in bacteria</p>
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<p>Attempts are on to create transgenic animals by direct micro-injection of DNA into ova or stem cells and produce insulin and other proteins in milk on a commercial scale. This technology is called Molecular Pharming (Producing pharmaceuticals using genetically modified plants or animals)</p>
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