a. Detail how protein expression will be induced in your chosen expression system

MSc Molecular Biology and Biotechnology Coursework

Section Three

You wish to over express the Q gene you have placed into the pETSHU plasmid for expression under the T7 promoter.

The translated sequence of the Q-gene is shown below alongside key protein characteristics:

MTAIIKEIVSRRIRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFE

DHNPPQLELIKPFCEDLDQWLSEDDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKSDPENEPFDEDQHTGR

TKV

Number of amino acids: 183

Molecular weight: 21500.28

Theoretical pI: ~6.06

Ext. coefficient    20650

a. Detail how protein expression will be induced in your chosen expression system.

b. Set out an experimental scheme to lyse and collect cytosolic fraction containing your target protein. Pay careful attention to the components of your lysis buffer.

In this example we will use the Protein Purification simulator (a free web app that can be downloaded to Apple and Android phones should you wish. http://www.agbooth.com/pp_java/).  In this example the Q-protein is number 10 and a 2D gel is shown of the protein mix with the target protein highlighted. The information in this gel will help you think of a purification scheme for this protein.

c. Using the information given about the protein and the simulation software detail the protein purification protocol that you will use to isolate the Q-protein. You will require a yield > 80% and >30 mg.

Method - state the method you will be using.

Mechanism - give a detailed description of how the method works.

Rational - demonstrate how the method has been applied to this protein.

NB - in protein purification you do not use the same method twice in a protocol.

What pH will the purification be performed at?

What is the charge on the protein at this pH?

d. Once isolated you will need to confirm that the protein has retained its biological activity. To do this perform at BLAST search using the either the DNA sequence or transcribed amino acid sequence to determine the class of protein and then identify a suitable enzyme assay.