separation and purification of proteins

fractionation of proteins in complex mixtures

A salt is added to a solution of macromolecules to a concentration just below the precipitation point of the protein of interest (green).

After centrifugation, the unwanted precipitated proteins (red) are discarded and more salt is added to the supernatant to a concentration sufficient to precipitate the proteins of interest.

After a second centrifugation, the desired protein (green) is recovered as a precipitate and the supernatant is discarded.

The salting out is based on a competition between the added salt ions and the dissolved solutes i.e. proteins, for molecules of solvent.

size exclusion chromatography

Separates proteins based on their molecular weight

• Larger proteins elute of before smaller proteins

• The agarose beads (gel) in the stationary phase are porous with small pores and channels permeating the beads

• Larger proteins do not enter the bead through these pores while the smaller ones do

• Larger proteins travel between the beads and elute off first while smaller proteins take longer to travel as they travel through the beads and travel more slowly

ion exchange chromatography

Separates ions and polar molecules based on their affinity to the ion exchanger

two types of ion exchange chromatography 

• Anion-exchange (column resin is positively charged attracting negatively charged proteins)

• Cation-exchange (column resin is negatively charged attracting positively charged proteins)

process

Proteins bind to moieties which are oppositely charged by forming ionic bonds to the insoluble stationary phase.

Cationic stationary phase is used to separate anions and an anionic stationary phase is used to separate cations.

Bound molecules eluted and collecting by running higher concentration of salt through the column or changing pH of the column

affinity chromatography

separates proteins based on the binding affinity for the desired protein on the column bead - affinity ligand 

process

• A ligand (black) is immobilised by covalently binding it to the chromatographic matrix

• A mixture of macromolecules (orange, blue and green) is added to the column

• Only certain molecules (orange) specifically bind to the ligand, the other components are washed through the column

protein A chromatography

The most applied affinity system for the purification of Staphylococcal protein A (SPA) and smaller ligands derived thereof.

The affinity between protein A and IgG was one of the first native interactions to be explored for the development of an affinity system for protein purification.

separation by size 

PAGE

sample buffer containing

• Sodium dodecyl sulphate (SDS) denatures proteins and confers a negative charge

• DTT/beta-mercaptoethanol: reducing agents

process

The sample is boiled for 5 minutes and loaded into wells in a vertical polyacrylamide gel. A current is passed through the gel. Small proteins migrate quicker through the gel, larger proteins are retarded towards the top of the gel.

electrophoresis

proteins are separated by size along an electrical field

polyacrylamide gel makes a lattice allowing

small proteins to travel faster than larger proteins

isoelectric point

(pI) the pH at which a particular molecule carries no electrical charge

molecule net charge is affected by

its pH of its surrounding environment

isoelectric focusing (IEF)

At a protein's pI it is immobile in an electrical field. If a mixture of proteins is electrophoresed through a solution or gel that has a stable pH gradient in which the pH smoothly increases from anode to cathode - each protein will migrate to its position on the pH gradient corresponding to its p

2D PAGE

IEF can be combined with SDS-PAGE in 2D electrophoresis - where proteins are subjected to IEF in one direction and then to SDS-PAGE in the perpendicular direction.

Highly valuable tool in proteomics - cataloguing all of a cell expressed proteins - proteome.

mass spectrometry

Protein of interest is digested (cut up) into peptide fragments with a protease i.e. trypsin, then dissolved in volatile solvent. Dry nitrogen gas promotes the evaporation of a solvent from charged droplets containing the protein of interest - leaves gas phase ions whose charge is due to the protonation of Arg and Lys residues.

determines 

mass to charge ration (m/z)

resulting mass spectrum

consists of a series of peaks corresponding to ions that differ by a single ionic charge and the mass of one proton

tandem mass spectrometry _ MS/MS

Peptides are separated by the first MS and one peptide is sent through a collision cell where it collides with helium atoms. This breaks the peptide into further fragments which are directed into a second MS that determines the m/z values of these smaller fragments.

X-ray crystallography

1. Precipitation of a pure preparation of the protein

2. Crystal formation - solid crystal of pure molecule

3. X-Ray diffraction through the crystal - reveals a pattern

4. Capture of the diffraction pattern and rendering by computer software into mathematical co-ordinates

5. Deposition of the crystal structure into the database

Cryogenic-electron microscopy (cryo-EM)

Produces 3D images in atomic detail:

• DNA, RNA, proteins, viruses, cells, etc

• Can also show molecule dynamics

Purified sample is added to a grid with tiny holes. Samples in the grid are rapidly frozen in liquid ethane (vitrification):

• Stops the movement of cellular components so the electron beam can get clear images

• Protects the molecule from the electron beams

• Prevents crystal formation

electron microscopy

an electron beam travels through the sample and casts a 2D shadows or projections of the molecules onto a detector

image processing and 3D model building

computer software groups together hundreds of 2D shadows taken from different angles and combines them into a 3D reconstruction of the molecule