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A young investigator would like to express and purify a newly discovered gene. She decides to clone the gene into a plasmid that contains the sequence for a hexahistidine tag. Plasmids that contain a sequence encoding a hexahistidine tag will produce a recombinant protein with an additional six histidine residues attached to the N (or C ) terminus. Her gene of interest will be expressed as an N ‑terminal fusion protein containing the six residue histidine tag (N−HIS6−Protein−C) . Order the steps for construction of the expression plasm

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Answer and Explanation:

The steps for construction of the expression plasma

  • STEP 1 : Introducing Notl and Hindlll confinement sites at the ends of the gene of interest by means of PCR
  • STEP 2 :Digest the plasmid and DNA encoding gene with the Notl and Hindol confinement endonucleuses.
  • STEP 3 : Ligate the cut plasmid and DNA fragment
  • STEP 4 : Change  the plasmid into E.coli for expression
  • STEP 5 : Clean the protein by utilizing a column of immobilized [tex]Ni^{+2}[/tex]

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