Recombinant protein expression: diagnosing why a construct will not express, and the variables worth changing in order

Diagnose, then change one thing at a time

1. Establish which failure you have. Look for the protein in the soluble fraction, the insoluble fraction and the whole lysate. Absent everywhere means expression or stability; present only in the pellet means folding; present and soluble but inactive means folding, modification or a missing cofactor. The remedies differ completely.
2. Construct boundaries are the highest-yield variable. Truncating to a domain, moving the start and stop a few residues, and removing disordered termini frequently converts an unexpressable construct into a well-behaved one. Screening several boundary variants in parallel is more productive than optimising one construct at length.
3. Codon usage and the untranslated region. Optimising codons for the host and removing structure in the region around the start codon addresses translation initiation and elongation problems. This is cheap to try given synthetic genes and is worth doing early in a bacterial host.
4. Lower the temperature and slow it down. Reducing induction temperature and inducer concentration slows synthesis and frequently converts inclusion bodies into soluble protein. It costs a little yield and is the single most reliable solubility remedy in bacterial expression.
5. Change host before you refold. Refolding from inclusion bodies is laborious and often low yielding. Strains engineered for disulphide formation or rare codons, or a move to insect or mammalian expression, usually give better material for less work if the budget allows.

Fusion partners and tags

Solubility-enhancing fusion partners genuinely help some proteins and simply carry an insoluble passenger for others. They also have to be removed, which adds a protease step, a separation and a yield loss, so test whether the protein remains soluble after cleavage before committing.

Position the affinity tag deliberately. A terminus buried in the fold makes purification hard, and a tag adjacent to an active site can reduce activity without preventing binding.

Knowing when to stop

Some proteins do not express well in any accessible system, and continuing is a choice about budget rather than a technical question. Set a decision point before starting and honour it.

Where the protein is commercially available at usable quality, buying it is frequently cheaper than a second round of optimisation, and that comparison is worth making explicitly rather than by default.

Common questions

Why is my recombinant protein not expressing?

First establish whether it is absent, insoluble or inactive, by looking in the soluble fraction, the pellet and the whole lysate. The three have different remedies and treating one as another wastes months.

What is the most effective change to try?

Construct boundaries. Truncating to a domain or trimming disordered termini converts many unexpressable constructs into well-behaved ones, and screening several variants in parallel beats optimising one at length.

How do I get soluble protein instead of inclusion bodies?

Lower the induction temperature and inducer concentration first, then consider a solubility fusion partner or a different host. Refolding from inclusion bodies is laborious and usually a last resort.

Should I use a solubility tag?

Test it, and test whether the protein stays soluble after the tag is cleaved. A fusion that keeps an insoluble passenger in solution only while attached has not solved the problem.

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