Protein Purification Strategies

Course: Methods in protein chemistryRahman M. Mahfuzur

2012/01/11SLU

To seperate a particular protin from all other proteins and cell components There are many types of proteins within an ogranism Other components: nuclic acids, charbohydrates, lopids, small moleculesA gieven protein could be 0.001-20% of total protein.

Objectives

Proteins purification varies from one purification step to multi- step of purifixcationsOften more than one purification step is necessary to reach the desired purity, or step can be repetead if sample is available. Successful and efficient protein purification depends on appropriate methods selection.Methods should be sequence in a logical manner,

what kinds of materials are available/handle?what has to be removed/ completely?what will be the use of final products?what are economical constraints?

Protein purification

Three phase purification strategies(CIPP)

The four parameters

Every technique offers a balance between resolution, capacity, speed and recovery

Capture

Initial purification of targetRapid isolation, stabilization, concentration

Intermediate purification

Further removal of bulk contaminants: other proteins, nucleic acids, endotoxins

and viruses.Purification and concentration.

Polishing

Final removal of remaining trace impurities or closely related substances to achieve high purity

Protein property Technique

Charge Ion exchange (IEX)

Specific ligand recognition (biospecifc or nonbiospecifc)

Affinity chromatography (AC)

Size Gel filtration (GF)

Hydrophobicity Hydrophobic interaction (HIC), Reversed phase (RPC)

Isoelectric point Chromatofocusing

Protein properties Vs Technique

Ion exchange chromatography (IEX)

separates proteins with differences in surface charge.based on the reversible interaction, charged protein Vs oppositely charged columnIf, pHb>PIP Neg. bind positively charged anion exchanger ; ex- MonoQ columnWhen, pHb<PIP Pos. bind negatively charged cation exchanger ; ex- MonoS column

IEX chromatogram

Affinity chromatography (AC)

On the basis of a reversible or specific interaction between target and a specifc ligandBiospecific: antibodies binding proteins,Non-biospecific: histidine binding protein bind to metal Ion; IMAC

AC chromatogram

Gel filtration chromatography (GF)

allow separation of proteins with differences in molecular sizeGF is a non-binding method0.5% to 2% of total column volume.

FG chromatogram

Hydrophobic interaction chromatography (HIC)

separates proteins with differences in hydrophobicity based on the reversible interaction between a protein and the hydrophobic surface of a chromatography medium

Technique Vs three phase

combinations of chromatographic steps

IEX-HIC-GF

Considered as a standard protocolIf nothing is known about the target protein use IEX-HIC-GF.both anion and cation exchange could be used to get different selectivities within the strategy.

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