A new investigator would like to express and purify a newly discovered protein. She decides to clone the gene into a plasmid that contains the sequence for a hexahistidine tag. Plasmids that contain a sequence for a hexahistidine tag will produce a recombinant protein with six extra histidine residues at the N- or C-terminus of the protein of interest.
Order the following steps for construction of the expression plasmid, expression and purification of the protein. The gene of interest will be introduced between the NotI and HindIII restriction sites.
Purify the protein using a column of immobilized Ni2+
Introduce NotI and HindIII rrestriction sites at the ends of the gene of interest via PCR
Digest the plasmid and DNA encoding the genes with NotI and HindIII restriction endonucleases
Ligate the cut plasmid and DNA fragment
Transform the plasmid into E. coli for expression
My answer; 1,3,4,2,5 right ?
Correct! The correct order of steps for construction of the expression plasmid, expression, and purification of the protein is:
1. Introduce NotI and HindIII restriction sites at the ends of the gene of interest via PCR.
2. Digest the plasmid and DNA encoding the genes with NotI and HindIII restriction endonucleases.
3. Ligate the cut plasmid and DNA fragment.
4. Transform the plasmid into E. coli for expression.
5. Purify the protein using a column of immobilized Ni2+.
By introducing the NotI and HindIII restriction sites at the ends of the gene of interest via PCR, you create compatible overhangs that can be used for subsequent cloning steps. Then, digesting the plasmid and DNA with NotI and HindIII restriction endonucleases allows for the targeted cutting of both the plasmid and the gene of interest.
After digestion, the cut plasmid and DNA are ligated together, joining the gene of interest to the plasmid. This recombinant plasmid is then introduced into E. coli cells through transformation, allowing for the expression of the protein of interest.
Finally, the expressed protein can be purified using a column of immobilized Ni2+. The hexahistidine tag on the recombinant protein allows for specific binding to nickel ions immobilized on the column, enabling purification.
Your answer is almost correct. Here is the correct order of the steps for construction of the expression plasmid, expression, and purification of the protein:
1. Introduce NotI and HindIII restriction sites at the ends of the gene of interest via PCR. This step is necessary to create compatible ends for subsequent steps.
2. Digest the plasmid and DNA encoding the genes with NotI and HindIII restriction endonucleases. This step allows for the plasmid and the gene of interest to be cut at specific sites.
3. Ligate the cut plasmid and DNA fragment. This step involves joining the cut plasmid and the DNA fragment together to create the expression plasmid.
4. Transform the plasmid into E. coli for expression. This step introduces the expression plasmid into E. coli bacteria, which will then produce the recombinant protein.
5. Purify the protein using a column of immobilized Ni2+. This step involves using a specific column to selectively bind and purify the protein of interest, which contains the hexahistidine tag.
So the correct order is: 1, 3, 4, 2, 5.