Protein expression service: scoping and staging the work

Staging the project

1. Feasibility before production. A short feasibility stage tests expression and solubility in a small number of constructs and hosts and ends with a decision rather than a delivery. It is the cheapest information you will buy on the project, and a provider unwilling to sell one is a provider proposing you fund their risk.
2. Construct and host screening. Screening varies the boundaries, the tag, the fusion partner and the host in parallel. The value is in breadth rather than depth, so ask how many variants the screen actually covers and how a winner is chosen, because a screen of two is not a screen.
3. Scale-up is a separate decision. What works in a small culture does not always survive scale-up, and a provider should say so. Price scale-up as its own stage with its own acceptance criteria rather than assuming the feasibility yield holds.
4. Cell bank and material ownership. Settle who owns the construct, the cell bank and the process, and where they are stored. A programme that will re-order should hold or be able to obtain the bank, or the second batch is a negotiation from a weak position.
5. Acceptance criteria in the contract. Write the quantity, the purity method and threshold, the activity assay and the endotoxin limit into the agreement. Acceptance disputes in this category are almost always about criteria that were discussed and never written down.

What a provider should carry, and what you should

A provider should carry execution risk: that the agreed process was run competently, the analytics were performed, and the material meets the written specification. You carry biology risk: that this protein can be made in this system at all.

Contracts that try to move biology risk to the provider are priced accordingly, and the premium is usually larger than the cost of a staged approach that finds the problem early.

Comparing custom protein expression quotes that look different

Providers quote by stage, by milestone or per unit of delivered protein, and the three are not comparable until you normalise them against the same specification and the same stop points. Ask each to price your own staging rather than presenting theirs.

Ask what happens if feasibility fails. A provider whose answer is clear and whose charge for that outcome is modest is telling you they expect to find out early, which is what you want.

tagged protein purification, and choosing the tag

The tag decides the purification and part of the biology. Histidine tags are small and purify on immobilised metal in any buffer, including denaturing ones. GST and MBP are large, aid solubility, and purify on their own affinity resins with gentle elution. FLAG and other epitope tags purify on an antibody resin at high cost and high specificity. Strep-tag II elutes under very mild conditions. After that the questions are the same for all of them: terminus, linker, whether a protease site is included, and whether the tag comes off before the protein is used.

bl21 de3 as the default expression host

The bl21 de3 strain carries an inducible T7 polymerase in its chromosome, which is what makes it the default host for a T7 promoter vector, and it is protease deficient, which helps an intact protein survive. Basal expression before induction is the problem with a toxic product, and the answers are a tighter variant, a lower temperature or a different promoter rather than more inducer.

bl21 without the lysogen, and when that is wanted

The plain bl21 strain carries no T7 polymerase, so a T7 vector will not express in it, which is exactly why it is used for cloning steps and for vectors driven by a bacterial promoter. It keeps the protease deficiency that makes the family useful. Choosing between the two is a question about the vector's promoter rather than about the protein being made.

dh5 alpha for the cloning half of the work

The dh5 alpha strain is a cloning host chosen for transformation efficiency and for mutations that stabilise inserts and improve plasmid quality. It is not an expression host, and using it as one is a common reason a T7 construct produces nothing. Most expression projects use two strains for that reason, and the construct is sequenced out of the cloning host before it moves.

e coli bl21 de3 named in full, and why the strain is chosen

Writing e coli bl21 de3 names the species and the chromosomal T7 polymerase together, which is the whole reason the strain is used with a T7 promoter vector, and its protease deficiency helps an intact product survive. Basal expression before induction is the difficulty with a toxic protein, answered by a tighter variant, a lower temperature or a different promoter rather than more inducer.

dh5a as the cloning host beside it

The strain written dh5a is the cloning host, chosen for transformation efficiency and for mutations that stabilise inserts and raise plasmid yield, and it carries no T7 polymerase so an expression construct produces nothing in it. Most projects therefore use two strains, and the construct is sequenced out of the cloning host before it moves to the expression one.

A lentiviral expression system for a mammalian product

A lentiviral expression system gives stable integration in dividing and non dividing cells, which is what makes it the route for a hard to transfect line, and the cost is a multi plasmid production step and a biosafety assessment. Titre by functional assay rather than particle count is what makes the dose reproducible, and insert size limits both packaging and titre.

protein purification columns and the order they run in

protein purification columns are chosen as a sequence rather than singly: an affinity capture first where a tag or a natural ligand exists, then ion exchange or hydrophobic interaction to remove what co-eluted, then size exclusion to polish and exchange buffer. Each step loses product, so a three step process with modest per step yield can end below half, which is why the capture step is where effort pays.

Common questions

What does a protein expression service include?

Typically construct design and synthesis, transformation or transfection, small-scale expression and solubility screening, scale-up, purification and the agreed analytics, delivered against a written specification.

How long does a protein expression project take?

It depends on the host and whether the protein expresses at all. Bacterial projects on a well-behaved target are short; mammalian expression, difficult targets and anything needing a stable line take substantially longer. Stage the contract so you learn which you have early.

Should I pay a fixed price or by stage?

By stage for anything uncertain. A fixed price for a difficult protein means the provider has priced the risk of failure into your quote whether or not it happens.

Who owns the cell bank?

Whatever the contract says, so read it. If you expect to re-order, ensure you own or can obtain the bank and the process description, otherwise the second batch is a sole-source negotiation.

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