Question

1. Explain the following Figure using 5-7 sentences that should include the purpose of LDH gene expression and whether the result shown in the figure was according to the expectation of the experiment. You may write on the blank side of this quiz (8 points) SPECIFIC ACTIVITY OF LDH IN IPTG SUPERNATANT 3.500 3.000 2.500 e 2.000 1.500 1.000 0.500 0.000 9 10 15 25 20 FRACTION NUMBER 2. Describe, in detail, the effect of IPTG on PET-LDH vector containing cell during the log phase of growth in suspension culture. (Hint: Draw a figure and show the organization of LDH gene and also show how IPTG turn on the expression of LDH gene). (7 points) 3. Explain the role of using two different plasmids called PCRScript and PET 28 in order to achieve the goal of LDH gene expression in E. coli cell. (8 points) 4. Explain the mechanism of His-tagged LDH and Ni-NTA column in the purification of LDH enzyme. (7 points)

Answer 1

1. Here, as the protein purification is increasing, concentration of protein ( fraction number) in total fraction volume is also increasing. So, specific activity of LDH ( activity per unit mass ) is also increasing. When fraction number reaches around 20, there is sudden increase in specific activity. So, some threshold fraction is required to achieve desirable activity in enzyme kinetics.

2. IPTG is an inducer which mimics allolactose in inducing the expression of lac operon genes. E.coli can uptake IPTG independent of permease. It will indice the expression of genes under lac operon.Here, LDH gene in plasmid . Mostly T7 polymerase gene is placed downstream lac promoter which will be induced by IPTG. Now, there will be expression of T7 RNA polymerase which will now express our LDH protein in plasmid. Thus IPTG will induce the expression of LDH.

3. By using 2 vectors, we can efficiently express our gene of interest in prokaryotic system.T7 polymerase can efficiently synthesis RNA in E.coli system. It is resistant to rifampicin also. So, 2 plasmids are used : one for T7 pol expression and one for our gene of interest.

IPTG INDUCTION T7 PROMOTERLDH GENE LAC PROMOTER T7 GENE Lac I Lac I BE3 pET

4.His tag proteins have 6 histidine residues at one of its ends. This histidine has affinity for Ni - NTA present in the column. At pH 8 or above, his will chelate the metal ion and binds to it. So, it is easy to purify the protein by this method.