Protein Separation and Purification

Methods rely on specific properties of proteinWhy purify a protein? Isolate

Allows:Analysis of the biological propertiesUnderstand its structure Study interactions

No single procedure can be used to isolate every protein

Exploit specific characteristics (structure or function) of theprotein. Different steps should exploit a different characteristic

Ensure method has little/no effect on function

Early steps involve releasing your protein from the cells(normally by homogenisation) and low resolving procedures toremove the bulk of the unwanted proteins

Lysozyme Sonicator French Press

Removing crude extract

Ammonium sulphate precipitation (40%)exploits changes in the solubility of proteins as consequence of achange in ionic strength (salt conc.) of the solution

At low salt, the solubility of a protein increases with saltconcentration, ‘SALTING IN’.

But as salt conc. (ionic strength) is increased further, the solubilityof the protein begins to decrease, until a point where the protein isprecipitated from solution, ‘SALTING OUT’.

Low salt

Optimal salt conc. for solubilityWeak attractive force

High SaltCompete for water

Debye-Huckel Theory

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Molarity of AmSO4

1 2 3

Log ofSolubility

g mL-1

Fibrinogen

Hemoglobin

Serumalbumin

Myoglobin

Ammonium sulphate precipitation

1M 2M 3M

AmSO4

The concept of a Column

Ion Exchange Chromatography (IEC)

Separates molecules based on their charge

The side-chain groups of some amino acids are ionizable,e.g., lysine, arginine, histidine, glutamic acid, aspartic acidas are the N-terminal amino and C-terminal carboxyl groups

Thus proteins are charge molecules and can have a different chargeat a given pH because they have different compositions of ionizableamino acids

For any given amphoteric protein, there will be a pH at whichits overall charge is 0

(No. of negative charges equals the No. of positive charges)

This is referred to as the ISOELECTRIC POINT (pI)or ISOTONIC POINT of the protein

At a pH above its pI a protein will have a net negative chargewhile At a pH below its pI a protein will have a net positive charge

IEC resins are made by covalently attaching Negatively or Positivelycharged functional groups to a solid support matrix to yield Cationor Anion exchangers, respectively

Negatively charged exchangers bind positively charged ions – cationsPositively charged exchangers bind negatively charged ions – anions

At a pH=pI of a protein, it will not bind to an ion exchange resin

When a charged molecule is applied to an exchanger of opposite charge, it is absorbed, while neutral ions or ions of the same charge are eluted in the void volume of the column (the volume that is not bound). The bound protein displaces the counterions

Adsorbed molecules are commonly eluted with salt (changes ionic strength of the column buffer) or pH, which change the affinities of the bound proteins for the exchanger

Gel Filtration Chromatography (GFC)

GFC (also Size Exclusion Chromatography, Molecular Sieve Chromatography or Molecular Exclusion Chromatography)

Separates molecules based on their size (& shape)

It can also be used to determine the size and molecular weight of a protein

Separation occurs due to the differential diffusion of various molecules into gel pores in a porous matrix. For protein purification, the matrix typically consists of porous beads (with pores of a specific size distribution) of an inert, highlyhydrated gel

Largest MW comes off first

Separation is due to exclusion or inclusion from the gel matrix

Small molecules diffuse into the gel pores, retarding their flow through the column, while large molecules do not enter the pores and are rapidly eluted from the column

Proteins elute from the column in order of decreasing molecular weight

Common gel matrices are dextran, agarose and polyacrylamide. These matrices are manufactured with different degrees of porosity, and thus can fractionate different size ranges of proteins

Other Purification Methods:Affinity Chromatography:

Separates molecules based on specific interactions between the protein of interest and the column matrixE.g. Antibodies which bind Protein

Enzyme which binds a co-enzyme or inhibitorA ligand is covalently bound to a solid matrix (usually agarose) which is then packed into a chromatography column

When a mixture containing the protein of interest is applied to the column, the desired protein is bound by the immobilised ligands, while all other proteins in the mixture, which should have no affinity for the ligand pass through and are discarded

Affinity chromatography(with HIS-tagged proteins)

Affinity chromatography can be performed using a number of different protein tags.

poly-hisitidine

The histidine tag is very short (6 His residues) Should not alter the conformation of the tagged proteinShould not be involved in artificial interactions.

The poly-his tag binds to a nickel chelate resin

Eluted by 1.0 M imidazole

List of Other Methods:

Hydrophobic Interaction Chromatography:-hydrophobic interactions under high salt

Chromatofocussing:-Fractionates based on a pH gradient generated in an ion-exchange column

HPLC:-Similar to IEF and GFC, but uses pressure to increase flow rates in columns with small particle size to increase resolution of peaks

Methods for Assessing Protein PuritySDS-PAGE – Commonest method, rapid and sensitiveSDSsodium dodecyl sulphatePAGEpolyacrylamide gel electrophoresis

Migration of a moleculein a electric field

Separates materialsbased on size

Isoelectric focusing (IEF) can also be used, which separates proteins by charge differences (induced by a pH gradient)

2D gel electrophoresis – Combination of SDS-PAGE and IEFSeparates by charge in the first dimension and then by size in the second dimension

Mass spectrometry

Protein PurificationObjectives Purity Stable Cost Time

Protein purification stepsExtraction

Cell breakage chemical physical

Debris removalstraining centrifugation filtration

SolubilizationInclusion bodies Urea

Inhibition of proteasesPreliminary concentration

ammonium sulfate precipitation ultrafiltration dialysis

Purification stepsIon exchange Affinity chromatography Gel filtration Preparative HPLC

Final stepCrystallization