You are planning to start a new biotechnology company producing a recombinant pr

Question

You are planning to start a new biotechnology company producing a recombinant protein KLV that stimulate antiviral responses. The cDNA encoding the protein (600 bp) and a bacterial protein expression vector containing an ampicillin resistance marker (see the figure on the right) are available. Please design the experiment to clone the KLV gene into the expression vector starting from the cDNA clone of the gene. How do you verify the identity of the cloned gene in the expression vector? The expression of the protein can be induced with IPTG using this cloning vector. Please explain how IPTG induces the expression of KLV. How do you detect whether the protein is expressed or not in bacteria cell lysate by western blot using an antibody against GST? How do you purify the GST fusion protein from the bacterial cell lysate?

Explanation / Answer

a. The primers for the KLV gene should have two restriction sites given in the vector to efficiently clone into the multiple cloning sites of the vector. The KLV gene should be cloned such that the restriction enzymes are present at 5' and 3' end of the sequence. The vector and sequence should be double digested to get the sticky end. Then both the components should be ligated to yield a recombinant plasmid having the KLV gene in the multiple cloning sites.

b. The verification of recombinant plasmid can be done in 2 ways. First transform the plasmid into competent E.coli bacteria and plate on an agar plate having ampicillin antibiotic. This could be used as resistance marker for screening the recombinant plasmids. The plasmids which have this resistance cassette will only survive on this plate. Secondly, isolate plasmids from the colonies survived on this plate and perform a double digestion using the restriction enzymes used to clone the KLV gene. If the gene of interest is present in the plasmid, then you will be able to see the insert release which can be visualised on the agarose gel electrophoresis.

c. pGEX vectors have a tac promoter which are induced by the lactose analog IPTG. pGEX vector also has a lacI site which produces a repressor protein that binds to the operator region of the tac promoter preventing the expression until induced by IPTG. When the KLV gene inserted into this vector, it is under the control of tac promoter of the pGEX vector. So when this is induced by IPTG, the KLV gene is overexpressed.

d. When the KLV gene is cloned into the pGEX vector's multiple cloning site, there is GST coding site upstream of this site. The KLV protein will be a fusion protein of GST and KLV. This protein can be readily detected using an anti-GST antibody on the western blot.

e. The GST-KLV fusion protein can be easily purified by affinity chromatography using a glutathione-Sepharose matrix under mild conditions. When the fusion protein is applied onto the column, the GST-KLV protein readily binds to the column and impurities are washed away by the washing buffer. Then this protein is eluted from the column using mild, non-denaturing conditions. The separation of the KLV protein from GST tag can be done using the thrombin enzyme which cleaves at the precission protease site.

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