Bacteria are simple and cost-effective hosts for the recombinant production of peptides and proteins. Peptides are conventionally defined as molecules consisting of 2–50 amino acids (Friedberg et al., 1947) while polypeptides composing of more than 100 amino acids are classified as proteins (Kim et al., 2015). Here, the term “pepteins” refer to peptides and small proteins made of 30–300 amino acids, a class of polypeptide chains whose importance in many biological functions and cellular processes was not recognized until recently (Su et al., 2013; Storz et al., 2014). Today, Escherichia coli-based expression systems are used successfully in many biopharmaceuticals productions (Walsh, 2018). Among other post translational modifications, disulfide bond formation plays a crucial role for folding into the native state and hereby often influences stability, function, and activity (Bulleid, 2012). Production of disulfide bonds containing peptide and protein-derived bioactive compounds, such as antimicrobial peptides, growth factors, antibody fragments or nanobodies have been difficult using E. coli-based expression systems as the cytoplasm of E. coli has a reducing environment, preventing the formation of disulfide bonds (Bessette et al., 1999; Gaciarz et al., 2017). Several systems and procedures have been developed to optimize the formation of cysteine bridges, including modified E. coli host cells for enhanced oxidative folding in the cytoplasm or the translocation to the oxidative environment of the E. coli periplasm (de Marco, 2009; Salinas et al., 2011; Berkmen, 2012; Lobstein et al., 2012; Manta et al., 2019). In both cases, native disulfide bond formation relies on the correct de novo disulfide bond formation or the subsequential isomerization to the correct isomer. Despite successful examples, many of such targets tend to accumulate as insoluble aggregates and are forming incomplete or wrong disulfide bonds, resulting in low overall production yields (Teilum et al., 1999; Bhatwa et al., 2021). The formation of inclusion bodies (IBs, insoluble protein aggregates) in E. coli is currently considered as obstacle since the refolding of unfolded IBs to functional proteins is regarded as inefficient. Advantages, such as high-titer expression, easy separation from host cell proteins, resistance to proteolytic degradation, the possibility to express toxic targets and others, however, have made inclusion bodies already a versatile strategy for diverse industrial applications (Singhvi et al., 2020; Bhatwa et al., 2021). To this end, we developed the production platform Numaswitch (Nguyen et al., 2021) that grants access to the described advantages of IBs and simultaneously solves the problem of the inefficient refolding step. Numaswitch is based on bifunctional Switchtag proteins which are derived from a family of exoproteins being present in Gram-negative bacteria (Linhartova et al., 2010).

The bifunctionality of Switchtags relies on Ca²⁺ binding domains, so called GG repeats that are characterized by non-apeptides with the consensus sequence GGXGXDXUX (X: any amino acid, U: large, hydrophobic amino acid), that bind Ca²⁺ with µM affinities (Lecher et al., 2012). GG repeats are typically part of type 1 secretion substrates (T1SS) i.e., the protein Hemolysin A (Kanonenberg et al., 2018). Inside the cytoplasm of E. coli (low Ca²⁺ concentration of ∼100 nM), Switchtags and T1SS are rather unfolded proteins and prone to aggregation. After secretion to the surrounding by a dedicated type 1 secretion complex, where the Ca²⁺ concentration is ∼ mM, the binding of Ca²⁺ converts T1SS into folded, stable, and functional proteins. Here, the applicability of the Switchtags for the production of four disulfide bond-containing pepteins was studied demonstrating that the Numaswitch approach is a powerful tool for folding of these biomolecules efficiently into the correct and functional conformation.

Pepteins are hard-to-be-produced biomolecules, and neither chemical synthesis nor available expression strategies are serving as reliable and efficient manufacturing strategies, in particular for disulfide-containing targets (Miranda and Alewood, 1999; Huang et al., 2012). The application of E. coli is hindered by i.e. the reducing environment of the cytoplasm and, despite important developments in this context, like improved oxidative folding or translocation into the periplasm, aggregation and the formation of wrong isomers are causing low production titers (Singhvi et al., 2020; Bhatwa et al., 2021). In this study, we successfully applied bifunctional Switchtag proteins to evaluate their potential to produce disulfide-containing pepteins.

Switchtag proteins induce the aggregation of pepteins as inclusion bodies with all beneficial features, like high expression titers, high initial purity grades, accessibility of toxic pepteins and the synthesis accuracy of ribosomes (>99.99%). After being extracted and solubilized, Switchtags assist in the refolding step taking place in a biochemical reaction containing Ca²⁺ at mM concentration. Here, the binding of Ca²⁺ to the GG repeats induces the quantitative refolding of Switchtags and water-soluble, stable Switchtag peptein fusions are formed. Switchtags act as solubility tags acting chaperon-like and preventing intermolecular interactions of fused pepteins. Aggregation and/or precipitation of fused pepteins are prevented and pepteins are kept soluble. This is important, since it allows the time-dependent folding of pepteins along the thermodynamical energy landscape reaching finally the native state. Switchtags enable the efficient refolding of unfolded pepteins being produced as inclusion bodies and assist in the folding step as bifunctional tag. In particular, for disulfide-containing targets, keeping them soluble and counteracting intermolecular interactions allows auto-oxidation and/or the application of redox shuffle systems allowing the formation of the correct isomer (lowest thermodynamic energy level).

Since proteogenic impurities being present in the IB samples are not efficiently refolded, the refolding step represents a purification step yielding highly pure Switchtag peptein fusions (see Figure 1, Lanes S compared to R). In addition, prior to refolding when the pepteins are still unfolded, the IBs can be washed by i.e., detergents or organic solvents removing process related impurities like endotoxins quantitatively (data not shown).

The presence of disulfide bonds in antimicrobial peptides, like DS-THA is often involved in modulating the activity (Ramamoorthy et al., 2006; Castro et al., 2011; Dash and Bhattacharjya, 2021). Furthermore, expression of antimicrobial peptides, like DS-THA, in recombinant systems is often difficult, due to the toxicity towards the expression hosts. The Switchtag approached, grants access to these kinds of disulfide bond containing compounds, where cytoplasmic expression usually is no option. Nanobodies, such as VHH, can be produced in E. coli, for example in combination with a SUMO tag (Wang et al., 2021). The refolding of functional VHH demonstrates that the Numaswitch approach is applicable in case of proteins with distinct, maybe even, complex conformations. In case of hEGF, where incomplete formation of disulfide bonds or the formation of wrong isoforms is a challenge, (Chang et al., 1995; Chang et al., 2001), Switchtags enable the production of fully-oxidized, functional molecules. Importantly, the application of Switchtags is not limited to dedicated E. coli strains or expression hosts as it can be regarded as biochemical toolbox.

If Switchtag peptein fusion proteins are expressed as IBs, the Numaswitch approach is applicable and the IBs can be efficiently and functionally refolded in the presence of Ca²⁺. Cytoplasmic expression of scFv or hEGF usually reaches production yields of up to 30 mg or 250 mg per liter fermentation broth, respectively, and rely on the use of solubility tags, redox shuffle system or systems like CyDisCo for production and disulfide formation (Ma et al., 2016; Shams et al., 2019; Kim et al., 2021). The application of shuffle strains with an oxidative cytoplasmic environment is especially hindered by slow growth rates and low titers in combination with limited upscalabilitly (Lobstein et al., 2012). The obtained production yields, to the best of our current knowledge, are the highest achieved so far.

Current efforts focus on the production of larger proteins (>300 aa) with complex tertiary structures (i.e. enzymes, antibodies) in industrial-relevant scales. However, first data indicate that refolding efficiencies, hence the production yields of target proteins decrease with larger protein sizes attached to the Switchtag clearly drawing out limitations of the Numaswitch technology (unpublished data). To addess this challenge we develop Switchtag proteins which can be used for these kind of proteins and act Ca²⁺-independent or in the presence of alternative ions (unpublished data). For this, alternative proteins of the huge RTX familiy are analyzed which comprises more than 1,000 members (Linhartova et al., 2010). Such Switchtags would not only grant access to a broader class of proteins, but also be applicable for candidates that are instable or non-functional in the presence of Ca²⁺ ions.

In this study the Numaswitch technology was successfully used to produce peptein candidates ranging from 50 to 265 amino acids clearly demonstrating its potential of being an efficient and universal platform for peptide (2−50 aa) (Nguyen et al., 2021) and peptein (30−300 aa) production. For the first time, the advantages of IBs get accessible by solving the major bottleneck; Switchtags ensure the efficient and reliable refolding of IBs into functional pepteins. Since the expression of IBs and the biochemical refolding step is reliable and efficient, production processes can be established within weeks making Numaswitch a versatile and scalable production platform compared to chemical synthesis and alternative bioprocesses.