Protein purification

Protein purification is often the step that turns a successful expression into a sample you can actually use. After expression in E. coli, yeast, insect cells or another system, the protein of interest has to be separated from host proteins, cell debris, nucleic acids, salts, detergents and aggregated forms. Genosphere Biotechnologies handles this step as part of its recombinant protein services, with a strategy matched to what the sample is for.

This page is for projects where purification is the central question: functional validation, immunisation, antigen production, a biochemical assay, screening, analytical work or preparing a research batch. It complements the recombinant protein service offer and helps you set out your needs before requesting a quote.

Purpose of purification

Purification is not defined by a purity percentage alone. The right protocol depends on the protein, the expression system, the amount expected, whether a tag is present, solubility, stability and the kind of experiment planned after delivery. A protein meant for immunisation, an enzyme assay, a protein-protein interaction study or an analytical standard does not always call for the same level of preparation.

The specification may include a purity target, a final buffer, a minimum concentration, a soluble or refolded form, keeping a cofactor, removing a tag or particular care with aggregates. When these points are not yet settled, Genosphere can help define them from the sequence and the intended application.

Purification strategy

The strategy usually begins with the construct: native or fusion protein, affinity tag, cleavage site, sensitive domains, theoretical size, isoelectric point and solubility conditions. These guide the choice of capture, wash, elution and polishing steps.

• Affinity purification when the protein carries a suitable tag, for example for a fast, selective capture.
• Ion-exchange chromatography when the protein’s charge allows contaminants to be separated efficiently.
• Size-exclusion chromatography to improve homogeneity, limit aggregates or prepare a sample for a sensitive analysis.
• Buffer adjustment to stabilise the protein and prepare the sample for the test planned after purification.

How these approaches combine depends on how the protein actually behaves. A highly expressed but insoluble protein, a membrane protein, a protein toxic to the host or one sensitive to oxidation may need a more gradual optimisation.

From expression to purified sample

Purification can be built into a full project of expression in E. coli, expression in yeast or production in a baculovirus/insect-cell system. In that case the expression system and the construct design are chosen from the start with purification in mind.

It can also come into a project already under way, for example when you supply a sequence, a plasmid, an expressed construct or a sample to be reworked. What is known about expression, solubility, previous yields and storage conditions then helps avoid unnecessary trials.

Quality control and delivery

Quality control depends on the project, but it may include a protein-profile analysis, an estimate of purity, a check of the apparent mass, concentration monitoring and a record of the final conditions. The aim is to deliver a sample you can make sense of, not just a tube: you should know which buffer the protein is in, how it was prepared and what limitations, if any, were observed.

For some uses an intermediate purity is enough to continue development. For others an additional polishing step is preferable. The quote sets out the options chosen, the associated checks and the delivery conditions for your project.

Information useful for a quote

For a protein purification request, the most useful details are the sequence or the expected mass, the expression system you want or have already used, any tag, the target amount, the purity level needed, the final buffer, the intended application and any difficulty already seen: low expression, inclusion bodies, proteolysis, aggregation, loss of activity or instability on storage.

These details separate a standard purification from a project that needs development work. They also avoid promising a generic protocol when the protein calls for a more specific approach.

Related services

If your need concerns immunoglobulins rather than recombinant proteins, see the page on antibody purification. For a recombinant protein, you can request a protein purification or protein expression quote, describing the sequence, the expression system and what the sample is for.