Question

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

Answer 1

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 $Ni^{+2}$