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 plasmid. (Note: The gene of interest will be inserted between the NotI and HindIII sites.) Step 1:
Step 2:
Step 3:
Step 4:
Step 5:
a. Introduce Notl and Hindlll restriction sites at the ends of the gene of interest via PCR.
b. Digest the plasmid and DNA encoding gene with the Notl and Hindlll restriction endonucleuses
c. Transform the plasmid into E. coli for expression
d. Purify the protein using a column of immobilized Ni^2+
e. Ligate the cut plasmid and DNA fragment