153l lab 2 - Jeong Keun Kim 304346888 Chem 153L Lab Report...

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Jeong Keun Kim 304346888 Chem 153L Lab Report 2 Induction and Analysis of His-tagged Fusion Protein Expression in E. coli Introduction The purpose of this lab was to produce and purify YqhD, alcohol dehydrogenase. To do this, we placed E. coli in a culture that contained pETDuet, a plasmid that contains the yqhD gene. The pETDuet plasmid also contains 6 Histidine residues. This hexa-histidine tag will later be used to purify the target protein through affinity chromatography. After the plasmid has been taken up by the bacteria, the over expression of the yqhD gene can take place. However, the actual expression of the protein, yqhD, is regulated by Lacl-regulated promoters. To induce expression, we added IPTG (isopropyl- thio-galacto-B-D-pyranoside) to induce the promoters and onset the expression of the protein. We let the cells incubate for various different times then place them into a protein assay. We assay for protein over-expression at different times to see what size protein is being over expressed. For example, if we load a gel with a cell that has been incubating for 1 hr with a cell that has been incubating for 3 hours; we can see which protein is being over- expressed because the over-expressed protein will create a darker line in the cell that was incubating longer.
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