Recombinant protein production planned from the assay backwards: how mammalian protein expression, insect protein expression system routes, insect cell protein expression specifically, membrane protein expression, a cell free protein expression kit and cell free protein production each suit a different molecule, why recombinant protein synthesis and recombinant protein manufacturers quote on milligrams of something you have not yet defined, when to purchase recombinant protein or buy recombinant protein instead of expressing it, how his tag protein purification, protein purification his tag workflows, histidine tag protein purification, his tag beads, gst tag protein purification, gst protein purification, a protein purification kit, magnetic beads for protein purification, protein purification beads, magnetic beads protein purification, protein purification systems and protein purification by hplc, hplc protein purification or hplc for protein purification differ in what they leave behind and what an hplc assay or hplc buy decision adds to the train, and what protein characterization, characterization of recombinant proteins, protein quantification and protein quantification assays have to report before the protein is usable

Planning the production

1. Start from what the protein has to do. A binding reagent, a structural target, an enzyme assay substrate and an immunogen impose completely different requirements on folding, modification, purity and quantity. Write those requirements down first, because they select the host rather than the other way round.
2. Choose the host from the modifications required. Bacterial expression is fastest and cheapest and gives no glycosylation and limited disulphide formation in the cytoplasm. Insect systems handle larger, more complex proteins with some modification. Mammalian expression gives human like processing and is the default for secreted and glycosylated products. Cell free systems suit toxic proteins and very fast turnarounds at small scale.
3. Design the tag and its removal together. An affinity tag makes purification simple and can change solubility, activity and crystallisation. Decide at design time whether it stays or goes, and if it goes, include the protease site and plan the second chromatography step that removes the protease and the cleaved tag.
4. Screen constructs and conditions in parallel. Boundaries, tag position, fusion partners, temperature and induction strength all change yield and solubility, and small parallel screens answer in a week what serial optimisation answers in a quarter. This is the single highest return activity in the whole workflow.
5. Build the purification around the contaminant that matters. Affinity capture is a start, not a purification. Add polishing steps chosen against what the assay cannot tolerate: aggregates, endotoxin, host proteins, nucleic acid or the tag itself. A single step preparation is fine for a pull down and inadequate for a potency assay.
6. Characterise before anyone uses the material. Identity, concentration by a method appropriate to the protein, purity, aggregation state and activity. Report all five with the lot. A protein delivered with a concentration and nothing else will be argued about for months.

Milligrams is the wrong unit to negotiate in

Quotations are written in milligrams because that is easy to price, and requirements are written in milligrams because that is what the quotation asked for. Neither says anything about folding, aggregation state, endotoxin or activity, which are what determine whether the material works.

Specify the acceptance criteria instead: purity by a named method, aggregate content by size exclusion, endotoxin below a stated limit, activity in a named assay. Then let the supplier tell you what quantity of that material costs.

Purification residues that break assays

Imidazole, reducing agents, glycerol, detergents and residual protease all travel with the protein and interfere with downstream measurements. Cell based assays are particularly sensitive, and endotoxin from bacterial expression produces responses that are frequently mistaken for the protein's own activity.

Decide the final buffer against the assay that will use the protein, and include a buffer exchange step rather than diluting the problem. Report the final formulation with the material.

Characterisation is what makes the lot reusable

A well characterised lot can be compared to the next one, which is what makes results across months comparable. Identity, purity, aggregation, concentration and activity, measured the same way each time, turn a preparation into a reagent.

Keep those five values with the lot number and the storage condition. Laboratories that do this stop repeating experiments because a new batch behaved differently, which is the most common hidden cost in this whole area.

Common questions

Why is my protein in inclusion bodies?

Usually expression faster than folding. Lower the temperature, reduce induction strength, try a solubility partner or move to a periplasmic or secreted route. Refolding is possible but adds a development project and a yield loss, so change the expression first.

Should the affinity tag be removed?

Remove it when the protein is used for structure, for activity measurements where the tag could interfere, or as an immunogen where the tag would dominate the response. Keep it for routine pull downs and immobilisation, where it is doing useful work.

How should concentration be measured?

By a method suited to the protein and stated with the result. Absorbance using a calculated extinction coefficient is accurate for proteins with aromatic residues and unreliable for those without; colorimetric assays are matrix sensitive and standard dependent. Say which was used.

When is buying the protein the better answer?

Whenever a catalogue product exists at the required purity and activity, and the quantity is modest. Expression is worth doing when the variant is unusual, the quantity is large, the material must be owned, or no supplier offers the form you need.

Browse by service class

• Compare Gene synthesis on advertised prices