Organic Chemistry / Chemical biology

Protein purification

Separating a protein from a cell: precipitation, dialysis, ion-exchange, size-exclusion and affinity chromatography, and checking purity.

Undergrad

In a nutshell

Before a protein can be studied, used as a drug or turned into a crystal, it has to be separated from the thousands of other proteins, nucleic acids and small molecules inside a cell. Protein purification does this in a sequence of steps, each exploiting a different property: how soluble the protein is in salt, its electric charge, its size, or its ability to grip a particular partner molecule. For example, scientists often attach a short tag of six histidines to a protein; the tag binds tightly to nickel ions on a column, so the target sticks while almost everything else washes straight through. After purification, a quick gel or mass spectrum checks that only one protein is left. The same principles are used at enormous scale to make insulin and antibody medicines.

Getting the protein out

Cells are broken open (lysis) by sonication, high-pressure homogenisation, detergents or lysozyme, then centrifuged to remove debris. Working cold, with protease inhibitors and a buffer at a suitable pH, limits degradation. The aim of every subsequent step is to raise specific activity (activity per mg of total protein) while keeping yield acceptable; a purification table tracks both, and the fold-purification is the ratio of specific activities.

Precipitation and dialysis

At high ionic strength, salts such as ammonium sulfate compete for water and strip the hydration shell from protein surfaces, so proteins aggregate and precipitate (salting out). Different proteins come out at different saturations, giving a crude fractionation. Proteins are also least soluble at their isoelectric point (pI), where net charge is zero. The excess salt is then removed by dialysis: the sample sits in a semipermeable membrane bag whose pores pass small ions but retain the protein, and repeated buffer changes exchange the solution.

Chromatography

Ion-exchange. Beads carry fixed charges: anion exchangers (e.g. DEAE or quaternary ammonium) bind negatively charged proteins, cation exchangers (carboxymethyl, sulfonate) bind positive ones. Choosing a buffer pH above the pI makes a protein anionic. Bound proteins are eluted with a rising salt gradient or a pH change, weakest binders first.
Size-exclusion (gel filtration). Porous beads let small molecules enter the pores and take a longer path, so large proteins elute first. Calibrated columns estimate native molecular mass and oligomeric state, and the method doubles as a gentle desalting step.
Affinity. A ligand that binds only the target (a substrate analogue, antibody or metal) is immobilised. In immobilised metal affinity chromatography, a His_6 tag coordinates Ni^{2+} held by a chelating resin; imidazole, which competes for the metal, elutes the protein. GST tags on glutathione resin work similarly. Affinity steps can give large purifications in a single pass.
Hydrophobic interaction and reversed-phase HPLC separate by surface hydrophobicity; reversed-phase is excellent for peptides but often denatures larger proteins.

A sensible sequence uses high-capacity, crude steps first (precipitation, ion exchange) and high-resolution, low-capacity ones last (size exclusion).

Checking purity

SDS-PAGE denatures proteins and coats them with negative charge, so they migrate according to mass; a single band at the expected size suggests purity. Mass spectrometry confirms the exact mass and can reveal truncations or modifications, UV absorbance at 280 nm (from Trp and Tyr) or a dye assay quantifies protein, and an activity assay shows the protein is still functional.

Key points

Track specific activity and yield at every step; fold-purification measures progress.
Ammonium sulfate salting out gives crude fractionation; dialysis removes the salt.
Ion exchange separates by charge relative to the pI; elution uses a salt or pH gradient.
In size-exclusion chromatography large proteins elute first.
Affinity tags (e.g. His_6 on Ni^{2+} resin, eluted with imidazole) give large single-step purifications; SDS-PAGE and MS check purity.

Check yourself

5 questions

1In size-exclusion chromatography, which proteins leave the column first?
Large proteins cannot enter the bead pores, so they travel a shorter path and elute first.
2What is dialysis mainly used for after an ammonium sulfate precipitation?
The membrane retains macromolecules but lets small ions equilibrate with fresh buffer.
3A protein has pI 5.5. To bind it to an anion-exchange column, which buffer pH is most suitable?
Above its pI the protein carries a net negative charge and binds the positively charged anion exchanger.
4Why is imidazole used to elute a His_6-tagged protein from a nickel column?
Imidazole is the histidine side-chain ring, so at high concentration it displaces the tag from the metal.
5During a purification, total protein falls from 1000 mg to 10 mg while total activity falls from 5000 to 2500 units. What is the fold-purification?
Specific activity rises from 5 to 250 units mg^{-1}, a 50-fold increase (with 50% yield).

Recent preprints on this topic

Loading…

Matched by keyword from ChemRxiv via Crossref. Not peer-reviewed; read critically.