Question

You have purchased a protease that can specifically cleave the Gln-Gly peptide bond of the GluAsp-Asp-Tyr –Asp-Gln-Gly sequence present in any protein (and it cleaves no other sequence found in proteins). Describe how you can use this protease (in combination with other purification method/s) to obtain pure protein A starting from the purified mixture of proteins A, B, and C you obtained in sub-question 2A. Assume that you will only need a very small amount of protease to cleave a large amount of its target protein, so the remaining protease is an insignificant impurity. (1 point) C. What specific kind of chromatography would you use to separate proteins B and C from a mixture of B and C obtained in sub-question 2B? (1 point)

Answer 1

It is given that the protease can specifically cleave Gln-Gly peptide bond in protein having Glu-Asp-Asp-Tyr–Asp-Gln-Gly sequence

Ans. In a mixture of protein A, B and C

by using protease we will be able to get the fragment of protein cleaved at Gln-Gly peptide bond in Glu-Asp-Asp-Tyr–Asp-Gln-Gly sequence. So we will be able to get a fragement of protein and some smaller peptides of that protein. Then we can do spectromentry to identify a protein from its peptide mass fingerprint. There are databases of the molecular weights of peptides that are generated by a specific protease. After this step, molecular weights of the parent protein and its proteolytic fragments will be helpful to search genome databases for any similarly sized protein with identical or similar peptide mass maps. In this way we will be able to diagnose the type and molecular weight of the protein.

After searching the database we will have an idea about the type of protein. This will help us in deciding the type of chromatography to separate the proteins.

First of all the we will perform SDS PAGE in order to get various bands of different sizes so that we can have a clear idea about the fragments available in the mixture of protein, and to target the desired protein whose idea we have got from the mass fingerprinting.

After this we will perform 2D gel electrophoresis to get the idea about its isoelectric focusing point.

After the above two step we will first perform Gel filtration chromatography to separate proteins on the basis of molecular weights.

Then we will perform ion exchange chromatography to separate the protein on the basis of the charge on the protein.

The fractions obtained will again subjected to SDS PAGE and mass fingerprinting to check whether we have purified our protein or not.

In this way we will be able to pure all the three proteins from the mixture step by step.