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

Explanation:

This process involves a molecular technique for the expression and purification of a gene of interest.

The first step is to digest the plasmid and DNA encoding gene with NotI and HindIII restriction enzymes. Restriction enzymes/endonucleases cut DNA chains at specific sites. After digestion, the NotI and HindIII restriction site is introduced to the gene of interest (via polymerase chain reaction, PCR) to allow for easy bonding with the plasmid DNA. After the sites have been created, the gene of interest/DNA fragment is then bonded/joined together with the cut plasmid DNA using a ligase (an enzyme that joins two DNA together). The new plasmid is then transformed into E. coli (which is a good cloning vector) for expression. After multiple copies of the plasmid has been produced by the E. coli, the proteins thereof are then extracted and purified using a column of immobilized Ni²⁺.

From the above, we can deduce that

Step 1: b

Step 2: a

Step 3: e

Step 4: c

Step 5: d

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