Question

Describe the roles of the following in the manufacture of a protein product: Plasmids, restriction enzymes, ligases, bacteria

Answer 1

The protein which we wish to clone in a bacteria for obtaining it in huge quantity is first selected. The the the protein coding gene is cut with a particular restriction enzyme from the genome. Then the plasmid is also cut with the same restriction enzyme. Then the plasmid and the protein coding gene is placed together with DNA ligase enzyme. As both plasmid and the protein coding gene has been cut with same restriction enzyme, there should be overhanging sites present in both. So DNA ligases can join them to construct a recombinant plasmid containing the protein coding gene of interest. The plasmid should also contain the promoter and other sequences required for protein expression. Then this recombinant plasmid is transformed into a suitable bacteria in which the plasmid will express the protein. The protein either secreted in the medium or it can be isolated after lysis of the cell. In either case the protein is purified by downstream procedures and is ready for use.