Biochemical Analysis 3.1
Description
The main purpose of this module is to introduce the learner to the concept of a recombinant protein and protein over-expression and to show the learner how to take a protein from an over-expressed state in a cell line/microorganism, through to a crudely purified state in preparation for chromatographic purification.
The module will provide the learner with an understanding and some practical experience of protein expression, monitoring of expression, protein recovery from cells, protein quantification and analysis and crude purification techniques. In addition, the learners will be introduced to some well-studied examples of protein-based biopharmaceutical products and how they are produced.
The practical aspect of this module will produce a recombinant protein from E. coli. They will monitor the growth of the cells and the expression of the protein in the cells. They will lyse the cells to recover the protein and analyse the crude extract to quantify protein content and activity using spectroscopic techniques. The crude extract will subsequently be prepared for chromatographic purification using one or a number of techniques including, precipitation, dialysis and/or ultrafiltration.
Learning Outcomes
Identify and discuss some well known recombinant proteins/biopharmaceutical products
Explain homologous and heterologous protein expression in various expression systems including E. coli, yeast, mammalian cell lines and insect cells (with particular emphasis on E. coli protein expression)
Grow E. coli cells for the purposes of recombinant protein production and theoretically maintain a mammalian, insect and yeast cell line for the purpose of recombinant protein expression
Explain the concept of a protein purification scheme and be able to design a basic purification scheme for a native and a recombinant protein
Analyse and quantify proteins using various methods
Detail how to monitor protein expression/protein purity and activity using SDS PAGE, Western blotting and protein activity assays
Purify a protein in preparation for chromatographic purification