Isopropyl -D-1-thiogalactopyranoside (IPTG) For Protein Expression – Essay Example
The paper "Isopropyl -D-1-thiogalactopyranoside (IPTG) For Protein Expression" is an outstanding example of a biology essay.
Isopropyl-β-D-Thiogalactopyranoside (IPTG) is normally utilised during cloning experiments in which induction β-galactosidase activity is necessary (Robinson 14). It involves a combinative action of Bluo-Gal or X-Gal in a blue-white selection of recombinant bacterial colonies which then results in lac operon induced expression in Escherichia coli (Lutz 814). Evidently, IPTG operates through binding on lacl repressor and thus altering its conformation. This consequently results in repression of the β-galactosidase coding gene lacZ. Recombinant proteins are supposed to be expressible in practical quantities so as to achieve in vitro protein characterizations (Lutz 814). To this end, genes are normally placed into expressible vectors like PET28 and PET21. These consequently change into E. Coli or its modified strains for expression. Subject to a convenient resistance to an OD600 of 0.6, the plasmid cells are then grown at 370 C in LB media. At this juncture, cooling of the medium takes place till it reaches the expression temperature and consequently induced by the IPTG inducing agent for 4 to 48 hours (Wall 258). Cell growth is normally catalyzed by higher temperatures and hence only a shorter expression time is needed. On the other hand, proper protein folding is catalyzed by lower expression temperatures and hence require a longer expression time. To this end temperatures ranging from 40C to 370C are utilized. Upon expression, the centrifugation process is used to harvest the cells and resuspended in a lysis buffer with 100mM phosphate at pH7.2-7.6. The sonication process is used to disrupt the resuspended. Finally, centrifugation is used in removing cellular debris before undertaking the protein purification process (Lutz 814).