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.
Please explain as well…
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