PHAR2811 Drug Design and Discovery Protein Purification 2020 PDF

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Summary

4. PROTEIN PURIFICATIONObjectives: To teach you the how to separate mixtures of proteins into individual polypeptides components. To give you experience in constructing a fractionation strategy. How this will be achieved:You must read the description of protein fractionation techniques in this manua...

Description

PHAR2811 Drug Design and Discovery: Protein Purification

4.1

4. PROTEIN PURIFICATION Objectives: •

To teach you the how to separate mixtures of proteins into individual

•

polypeptides components. To give you experience in constructing a fractionation strategy.

How this will be achieved: You must read the description of protein fractionation techniques in this manual. You will be given a solution which contains a mixture of 3 proteins. It is your task to separate that mixture into the three, separate pure proteins and to identify each protein. You will be assessed on the effectiveness of your fractionation and on the process (ie, the logic) by which you arrived at your assignations

Why do All This? Protein fractionation is one of the methods used to purify a protein of interest and is an exceptionally common technique in Molecular Biology and Biotechnology. Before we can obtain information about the shape or mechanism of action of a protein, it must be purified. Any protein product made by recombinant DNA technology still has to be separated from the contents of the cell it was expressed in. Similarly, drugs once synthesised, must be purified and characterised. If proteins are to be used as pharmaceuticals then one must know how to purify and characterize these. Many of the separation and purification methods used are based on the physical and chemical properties of molecules; chromatographic separation being common to both proteins and drugs.

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PHAR2811 Drug Design and Discovery: Protein Purification

4.2

SAFETY Task

Hazard

Associated harm

Existing risk control

Current risk

Additional controls required

Residual risk

SDS PAGE

Working with 0.1% SDS

Skin contact

Use of PPE (gloves, labcoat)

1 (low)

no

Pre-cast gels so contact minimised

Skin contact

Use of PPE (gloves, labcoat), demonstrator to set up gels and stain.

1 (low)

no

Pre-cast gels so unpolymerised acrylamide residual would be very small

Electrocution

Supervision by demonstrator, equipment designed to minimise contact with buffer when power is on

1 (low)

no

Electrocution

Supervision by demonstrator, equipment designed to minimise contact with buffer when power is on

1 (low)

no

SDS PAGE

SDS PAGE

Zone electrophoresis

unpolymerised acrylamide

Electrophoresis

Electrophoresis

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PHAR2811 Drug Design and Discovery: Protein Purification

4.3

1. INTRODUCTION Each pair will be given a mixture of three proteins. This mixture will be unique to your group. Your mixture will contain between 4 and 8 mg of any three of the following proteins: Myoglobin, Haemoglobin, Cytochrome c, α-lactalbumin, Ribonuclease, Bovine Serum Albumin, Ferritin and Catalase. It is your task to separate and identify these three proteins. It is your responsibility to

1.1 Information that will help you identify your proteins

come prepared for this practical session. Otherwise it could be a very long day!

The mixture of three proteins given to you will contain either 2 heavy and 1 light protein or 1 heavy and 2 light proteins. For the purposes of this experiment, a heavy protein is defined as one with a molecular weight of over 50,000 and a light protein is one with a molecular weight of less than 50,000. The two smaller/heavier proteins in the mixture have pI’s that differ by at least 2 pH units. These can be separated by ion exchange chromatography (charge) at pH 7.5.

1.2 Before coming to class… 



Read through the description of the different protein separation techniques available and examine the properties of the different proteins that could be in the mixtures (see appendices at the end of this section). Construct a table in your notebook to summarize the information. Such a table will be invaluable in designing your strategy. For example:

Protein

Lysozyme

Mol. Wt. 14,300 How will it behave on a G-50 column? It will be retarded because Mw is pI the protein is in a relatively basic environment and it becomes -vely charged, therefore it migrates to the anode (+ve end).

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PHAR2811 Drug Design and Discovery: Protein Purification

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A scan of an example Zone Electrophoresis Gel is shown below. Positive proteins (+) will move to the cathode (-ve). These proteins have pIs >8.8

Cathode (-ve) 1

2

3

4

5

6

origin

7

8

origin

+

Negative proteins (-) will move to the anode (+ve). These proteins have pIs...