Solved: Heterologous Gene Expression Problems

When heterologous gene expression goes wrong it can be a real headache. Here’s my checklist for the steps to take when you encounter problems with this dark art.

1. Check the construct by sequencing the expression cassette to make sure that everything is as you expect. A lack of expression could result from a stray stop codon.

2. If possible, don’t rely on SDS-PAGE with Coomassie staining as your only assay for expression. This is a relatively insensitive technique and your protein could be expressed even if there is no band present. If possible, use a western blot or activity assay to verify whether you have expression.

3. Check whether your expressed protein is soluble. A good SDS-PAGE/Coomassie band does not necessarily mean that all is well. The band could be insoluble, non-functional protein. To check this, prepare the sample as normal but after lysis, centrifuge the cells at maximum speed. Collect the supernatant (this is the soluble fraction) then re-suspend the pellet in fresh buffer to the same volume as the supernatant to get the insoluble fraction. If your protein is in the insoluble fraction it means that it is not folding properly, which is almost as bad as having no expression at all.

4. Try a different promoter. Some promoter/gene combinations just don’t work because of secondary structure formation between the 5′ untranslated region and the beginning of the coding sequence, which prevents the ribosome from efficiently translating the mRNA. If you get no expression at all, it is a good idea to try another promoter.

5. Slow things down. If your protein is expressed too quickly the cell may unable to cope with folding the protein, resulting in insoluble expression. Lowering the growth temperature or reducing the inducer concentration will theoretically slow down the expression and might allow the folding apparatus to keep up. For for IPTG induction, you could try Molecula’s Inducer as an alternative to IPTG.

6. Co-express a chaperone. If slowing things down doesn’t help, then increasing the cell’s capacity to fold proteins might. Heating the culture to 42°C or adding ethanol (to around 3% final conc) an hour or so before induction of gene expression increases the expression of endogenous chaperones. Also, kits such as Takara’s Chaperone Plasmid Set allow you to over-express specific chaperones.

7. Soluble fusion proteins can help overcome problems like both lack of expression and insoluble expression. Proteins like maltose binding protein and thioredoxin are themselves solubly expressed to very high levels, so can drive the same type of expression for proteins fused to them. Try both N and C-terminal fusions and make sure that the function of your enzyme is retained.

8. Fix the codon usage. Check that the codon usage of your heterologously expressed gene fits reasonably well with the host (more info here). If E.coli is your expression host, switching to Stragene’s Rosetta strain, which carries extra copies of the tRNA’s that are rare in E.coli, could help. Otherwise site directed mutagenesis or whole gene synthesis may be required.

9. Help disulfide bond formation. Some proteins require disulfide bonds between pairs of cysteine residues for proper folding. E.coli strains like Stratagene’s Origami have mutations that assist disulfide bond formation and may help in these cases.

10. Try another expression system. Some proteins just can’t be expressed in a given expression system. If all else fails, switching to an alternative expression host that is more similar to the source of the heterologous gene could be the answer.

Heterologous expression is by no means an exact science. The best you can do is to try as many options as possible and hope that something works. If you have any other suggestions or questions on how to approach problems with heterologous gene expression feel free to leave a comment.