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Continuing our look at strategies for handling expression of troublesome proteins, this post is the second part in our a series on protein expression, purification, crystallization, and analysis. If you missed the first part, you can find it here.

In this brief overview, we will look at issues related to physical and structural characteristics of the protein, as well as issues rising from the coding sequence itself.

Mis-folding proteins

The proper formation of disulfide bonds is often key to correct protein folding and thus for the end product to be functional. At the same time, it can also help avoid the formation of inclusion bodies from aggregated peptides, or premature degradation of mis-formed peptide.

Heterologous protein expression in E. coli can be negatively impacted by the normal redox state within the E. coli cytoplasm. A common work-around has therefore been to export the protein of interest to the periplasm, where the environment is more oxidative, and disulfide bond (Dsb) proteins can act on the peptide to ensure proper disulfide bond formation. This can be accomplished by the inclusion of an N-terminal signal sequence in the expression vector to direct the product to the periplasm or by fusing the peptide of interest to a wild-type maltose-binding protein containing such a signal.

Alternatively, direct alteration of the cytoplasmic redox environment by genetic engineering of E. coli, sometimes along with expression of cytoplasmic Dsb proteins to further assist correct protein folding, can be used, as is the case in e.g. SHuffle expression strains.

The aforementioned pMALp5 vectors also have a use when attempting expression of proteins with a known low-solubility. The addition of a MBP-fusion tag to a low-soluble protein are usually not expressible, even when taking measures such as tunable expression (discussed in the first part of this series), but require other tweaks to the expression strategy.

In some cases, a lower temperature may increase yield(NEB, unpublished data), however changing the expression vector to include an in-frame MBP fusion tag may work better as it can aid not only expression, but also solubility, and allow for easier downstream purification of the protein of interest from the host cell lysate using an amylose column. In many cases, analysis is possible directly after this. Otherwise the MBP tag can be removed by directed proteolytic cleavage, and the protein of interest isolated.

A third strategy for improving target protein solubility is to co-express chaperonins, e.g. DnaK or GroEL, with the caveat that while chaperonin overexpression may help alleviate the issue of target protein solubility, it may also result in chaperonin-target complexes which, depending on the workflow, will need to be further adressed before final analysis.

Membrane protein issues

Apart from direct folding issues, incorrect targeting of proteins may also cause problems. For membrane proteins in particular a common practice has been to target them to the E. coli inner membrane which, in cases where constitutive expression is employed, may cause the SecYEG translocation pathway to become blocked with freshly synthesized peptides. To avoid this, one can utilize the previously mentioned Lemo21(DE3) Competent E. coli, which, with its variably inducible promoters can allow for expression to be tuned low enough to avoid congestion.

Researchers at New England Biolabs have also found that problems with expression of difficult membrane proteins can be overcome by shifting from a high-copy number plasmid to a low- or medium copy plasmid. Changing the resistance gene in use, e.g. from ampicillin to kanamycin or chloramphenicol, may also enhance yield by ensuring a higher degree of plasmid retention(NEB, unpublished data).

When employing this strategy, NEB recommends confirming the presence of the expression plasmid at the point of induction, and also to check for target protein membrane integration after the experiment. If further problems are encountered, it is recommended to also lower the cultivation temperature of the cells to 20-25 C to promote membrane insertion and induce cells already at an OD of between 0.35-0.45.

Coding sequence issues

The coding sequence itself can cause issues when expressing heterologous proteins. Direct transfer of a coding sequence from one organism to E. coli may lead to issues with impaired expression, as the transgenic sequence can utilize codons for which the corresponding tRNAs are not abundant enough in the host cell, leading to slow, or even halted, translation.

Additionally, certain nucleotide sequences can result in the formation of secondary structures which adversely affect or even prohibit translation. Issues casused by the secondary structure formation are most commonly encoutered within the 5’ UTR, the coding region of affinity tag(s), or the ribosomal binding site. In such cases, translation can may be improved by altering the ribosomal binding site to better correspond to the optimal E. coli specific sequence of “AGGAGGT”, while issues relating to an affinity tag may require replacing it with an alternate tag, or moving the tag to another part of the peptide.

In cases where tRNA abundance disrupts translation, two main strategies have been presented: The first is co-expression of the relevant tRNA(s) in the host strain and, the second, a complete re-design of the coding sequence to better suit the host organism’s endogenously available tRNAs. However, since there’s no such thing as a perfect world – both of these strategies run the risk of causing expression to become so successful that it results in downstream problems e.g. causing inclusion bodies or overloading translocation machinery, as discussed earleir. Therefore, it may be wise to couple any re-designed gene to an inducuble system from the start to ensure that the host strain is not adversely affected.

Sometimes though, even all the optimization in the world won’t work for expressing proteins in host cell systems. That’s when cell free expression can be the best choice, but there will be more on that in a later post!

Until then, if you have any issues with your protein production, always feel free to reach out to us, we’re happy to help.