Clary Leonhart used the pET vector system to express her prokaryotic amylase enzyme. added peptone into her culture broth of BL21(DE3) Escherichia coli strain. At the end of the experiment, she discovered that her protein was not expressed. She repeated three more times but her protein of interest was still not produced. (i) (ii) Explain the reason why Clary failed to obtain her protein of interest and suggest a solution to troubleshoot this problem. Clary plans to express her protein along with a polyhistidine-tag, or better known by its trademarked name IIis-tag. Explain the importance of His-tag in protein work.
i) Clary failed to obtain her protein of interest in this experiment because she forgot to induce protein expression with IPTG (Isopropyl β- d-1-thiogalactopyranoside).
pET vectors are used for the overexpression of any protein in bacteria.
The pET vector contains a T7 promoter, which can turn on the expression of the gene of our interest with T7 RNA polymerase. The host system (BL21) is engineered to carry the T7 RNA polymerase gene under the control of the lacUV5 promoter. Expression of the T7 polymerase is induced by adding the lactose analogue IPTG to the bacterial culture. The pET vector contains the T7 promoter fused with the lac operator (lacO) and natural promoter and the coding sequence for lacI. In the absence of induction, lacI Inhibits the expression of both genes of interest and T7 RNA polymerase. To induce the expression, IPTG, which is a lactose analogue, is added, and IPTG binding to lacI repressor results in the expression of Host T7 RNA polymerase, which can act on T7 promoter, which was earlier unavailable due to lacI repressor binding and fire up the expression of the gene of our interest.
To troubleshoot this problem, Clary has to add varying concentrations of IPTG and see whether the protein is getting induced or not.
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