It is well known that expression of many mammalian genes in E. coli can be challenging and lead to low titers. This can be due to various reasons, including rapid degradation of the protein after inclusion body formation, toxicity issues resulting in low growth rate, and aggregation of the protein resulting in complexes with aberrant biological activity.

Two other reasons are sub-optimal translation of the gene and rapid degradation of its mRNA. It is hypothesized that these phenomena can be avoided by fusing sequences to the 5' end of the coding part of the mammalian gene. Whether this ensures more efficient translation initiation because the ribosome is presented with a different 5' coding sequence, because it protects the 5' end of the mRNA from degradation, or other more cryptic effects, or combinations thereof, is unclear.

In Vectron we regularly use two different types of 5' fusion partners for elevating the expression titers (Figure 2): Signal peptide sequences (SP) when the target proteins is to be transported to the periplasmic space, and our proprietary CYT tag sequences when the protein is to remain the cytoplasmic space.

Figure 2: Constructs that contain N-terminal fusion partners for elevated titers. The partners are either signal peptides (SP) or CYT partners (CYT). The former will in addition to typically increasing titers, result in the protein being transported to the periplasm. In either case an efficient cleaving site must be part of the SP (resulting in cleaving as the protein traverses the periplasmic membrane) or positioned between the fusion partner and the target protein, allowing for downstream processing where the CYT partner is efficiently removed without leaving any non-native amino acids.

We have previously described the use of signal peptides for increased translocation and expression, demonstrating that in addition to directing the protein to the periplasmic space, signal peptides can also strongly elevate expression rates (Sletta et al, 2009), and that it is possible to engineer signal peptides for improved functionalities (Heggeset et al, 2014).

We are currently using signal peptides for both the Sec pathway and the Tat pathway and will for each protein target determine which pathway and which signal peptide is the most suitable choice. This can not be done without a screening of multiple potential signal peptides, including native signal peptides and our proprietary, engineered signal peptides.

Figure 3: Schematic overview of E. coli translocation to the periplasmic space using signal peptides.

In some cases it is not an objective to have the protein transported to the periplasmic space, and hence signal peptides are unsuitable fusion partners to use for increasing the production titers.

Through the development of Vectron's technologies, fusion partners that has the potential to greatly affect cytoplasmic titers when fused to the 5' end of recombinant genes, have been developed. This are small partners that increase expression without affecting folding. We have named these fusion partners the CYT tags.

One particular CYT tag, the CYT2 tag, seems to be particularly efficient at increasing the titers of many different genes, as can be seen from Figure 4 below. The effect can be substantial. For some genes without a CYT fusion partner, including GM-CSF and G-CSF, the titers is non-detectable, whereas with CYT partners, strong bands are observed.

Figure 4: The effect of Vectron's CYT fusion partners for increased expression of some therapeutic proteins. For GM-CSF and G-CSF the effect is absolute, increasing the titers from non-detectable to a strong band on Western Blot. For TNF-alpha1A the effect is substantial, and increases the titers many-fold.

We are currently applying the CYT technology to customer-proteins and expect to present more data in future newsletters.

To conclude, using 5' fusion partners can greatly increase titers of proteins produced in both the periplasmic space and the cytoplasmic space, and is thus suitable for both soluble and insoluble proteins. It is not possible to predict which signal peptide is ideal for each and every protein, and a screening must be performed to identify the one that is optimal in each and every case.

Jostein Malmo, CSO
"The CYT technology is a valuable addition to our expanding vector toolbox. So far, we have seen a considerable increase in both soluble and total protein for the proteins where this fusion partner was tested. By choosing an appropriate cleavage strategy, the fusion partner can be efficiently removed downstream without leaving any nonnative amino acid residues."

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Bibliography

Sletta, H., Tøndervik, A., Hakvåg, S., Aune, T. E. V., Nedal, A., Aune, R., Evensen, G., Valla, S., Ellingsen, T. E., and Brautaset, T. The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli. Applied and Environmental Microbiology. Feb 2007, p. 906-12.

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Heggeset, T. M. B., Kucharove, V., Nærdal, I., Valla, S., Sletta, H., Ellingsen, T. E., and Brautaset, T. Combinatorial Mutagenesis and Selection of Improved Signal Sequences and Their Application for High-Levek Production of Translocated Heterologous Proteins in Escherichia coli. Applied and Environmental Biology. Jan 2014, p. 559-68.