Question

You recently joined a biochemistry research lab to study protein functions in diseases. In the lab, researchers have been working on a protein kinase, the enzyme that catalyzes the phosphoryl transfer reaction from ATP to the -OH group on serine or threonine residues of the target proteins. The researchers in the lab have found that a point mutation in the gene encoding the protein kinase leads to a change in one amino acid of the enzyme, which causes an increase in the kinase activity. They have also found that mouse models with the mutated gene are 50 times more likely to develop cancer than the wildtype animals. However, they have yet to find out what mutation leads to the abnormal activity of the enzyme.

In the lab, you are tasked to work with some other lab members to find out what the mutation is. One lab member suggests that you run an SDS-PAGE of both wildtype and mutated enzymes to find out what mutation occurs in the protein. Can you explain if SDS-PAGE can provide information on the mutation in the enzyme? Why or why not? If you think this is a good suggestion, describe briefly how you will use this technique to figure out the mutation. Make sure to describe what materials you will need to do the experiment, and provide a brief procedure.

Answer 1

SDS-PAGE is the basic technique one can use here. There might be possibility that mutation can affect the length of polypeptide (protein kinase enzyme). On SDS-PAGE, one can clearly distinguish the molecular weight of wild type as well as mutated protein by comparing with molecular weight standard. In my opinion, in-silico approach should be used to identify the mutation. SDS-PAGE can only tell difference between the molecular weight of mutated and wild type protein.

In-silico approach involves knowledge of bio-informatics. Uniprot, PDB,Ensembl, OMIM or dbSNP are the few known approches one can utilized in identifying the mutation.