A young investigator would like to express and purify a newly discovered gene. S

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 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:

The options are:

-Introduce Notl and Hindlll restriction sites at the ends of the gene of interest via PCR.

-Digest the plasmid and DNA encoding gene with the Notl and Hindlll restriction endonucleuses

-Transform the plasmid into E.coli for expression

-Purify the protein using a column of immobilized Ni^2+

-Ligate the cut plasmid and DNA fragment

Explanation / Answer

Step 1:Introduce Notl and Hindlll restriction sites at the ends of the gene of interest via PCR.

Step 2:Digest the plasmid and DNA encoding gene with the Notl and Hindlll restriction endonucleuses

Step 3: Ligate the cut plasmid and DNA fragment

Step 4: Transform the plasmid into E.coli for expression

Step 5:Purify the protein using a column of immobilized Ni^2+

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.