04 Protein Purification

04 Protein Purification - Protein Purification the problem...

Info iconThis preview shows pages 1–6. Sign up to view the full content.

View Full Document Right Arrow Icon
Protein Purification – the problem It is often necessary to purify a protein from the cell in which it is produced. This is not easy, since the cell contains hundreds of different proteins, and it is unlikely that the protein to be purified will make up more than 1 – 2% of the total cellular protein. Most likely it will make up less than 0.1%. The other proteins which surround the protein to be purified all have similar properties. They are not robust enough to withstand extreme conditions, and easily lose the shape on which their properties depend ie become denatured . They must be handled at close to neutral pH, and not exposed to high temperatures.
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
The Approaches to a Solution The general solution to this problem is to look for ways in which the protein’s properties differ from other proteins. This may be in: Its electrical charge. Its size. Hydrophobic residues on its surface. Its binding (recognition) properties. Its solubility in salt solutions. Its temperature stability. It is most likely that, unless the protein is very unusual in some way, a combination of methods will have to be used.
Background image of page 2
Production of a Cell Extract Before a purification procedure can be started an aqueous extract containing the protein must be produced. If the protein is cellular it will be necessary to break up the cells, releasing their contents. For cells with a tough cell wall, like bacteria or yeast, this will require a pressure cell such as a French press. Animal tissue can be homogenised using a Potter- Elvejhem homogeniser, or a blender. Plant tissue will require a blender to break its tough cell walls. In all cases the homogenate produced must be centrifuged to remove cell wall material, connective tissue etc.
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Crude Initial Stage Methods The first purification step will normally involve a method such as fractional precipitation with increasing concentrations of ammonium sulphate (salting out), or heat denaturation. These methods are not designed to achieve total purification, but to remove as much contaminant protein as possible whilst retaining all, or nearly all, of the protein of interest.
Background image of page 4
Ammonium Sulphate Precipitation Most proteins will precipitate from solution at high salt concentrations, but the salt concentration required to precipitate them varies considerably. If the salt concentration required to precipitate the protein of interest is known, proteins which precipitate at lower concentrations can first be removed and discarded. A fraction containing the protein to be purified can then be precipitated and collected, leaving behind any protein which is still soluble. Proteins precipitate over quite a wide range of salt concentrations so
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 6
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 17

04 Protein Purification - Protein Purification the problem...

This preview shows document pages 1 - 6. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online