At a Glance
In bacteria, protein secretion is mainly orchestrated by the Sec Pathway via Signal Peptides (SP), which are located at the N-terminus of secreted proteins. The secretion efficiency is not determined by the sequence of the SP alone, but instead is the combined result of an SP with its specific target protein. This necessitates establishing efficient screening procedures to evaluate all possible SP/target protein combinations. We developed such an approach for our Signal Peptide Toolbox, which contains 74 Sec-dependent SPs. It combines combinatorial construction with highly reproducible, quantitative measurements. By applying this procedure, we demonstrated the secretion of three different proteins and succeeded in identifying the most potent SP-protein combination for each of them. This thoroughly evaluated measurement tool, in combination with our SP toolbox (fully available via the partsregistry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest.
Over the course of the last decades the quality, amount and spectrum of heterologous (and recombinant) proteins has drastically increased and therefore the need for techniques to easily express and purify these proteins has emerged. We find such proteins as ingredients of detergents (proteases), medical treatments (insulin) or food and beverage products (amylases). Simply put, heterologous proteins are ubiquitously present. 
In order to tackle this demand we chose to apply the genetic tools of the model organism Bacillus subtilis. It is already one of the most frequently used hosts for overproduction of proteins throughout academia and industry because of its tremendous capacity to secret proteins, which can be exploited to increase the overall yield.
B. subtilis has four different secretion pathways, however the majority of proteins are being secreted via the general Sec pathway (Figure 1). This pathway has been identified in playing a crucial role in protein secretion as a common element among all domains of life . In the Sec pathway, the secretion of proteins into the surrounding supernatant is orchestrated by signal peptides (SP). These SPs are composed of approximately 60 to 180 nucleotides and they are N-terminally attached to the protein thereby orchestrating the secretion. Intracellularly, the SP is translationally fused to the specific protein but cut off during the membrane translocation process releasing the protein into the supernatant without the signal peptide attached to it. 
Bacillus subtilis contains approximately 170 Sec Signal Peptides (SPs) that re-direct proteins for secretion via the Sec pathway. For our toolbox we were able to clone 74 Sec SPs (Table 1). Each SP was amplified from genomic DNA of B. subtilis wild type with the primers found in the primer collection table at the end of the Design section. After amplification, each SP was digested using the restriction enzymes EcoRI and PstI, stored into the pSB1C3 backbone and submitted to the partsregistry.
This powerful collection of SPs can now be combined with our Evaluation Vector and any protein of interest (POI) for a shotgun cloning approach to identify the best combination of SP and POI.
Though having access to these SPs via the registry, does not solve the problem having to create one clone for each single SP and POI. This also means, sufficient amounts of each SP are necessary to guarantee efficient cloning. Thus, we followed up on the idea of multi-template PCR. Since all SPs are stored in the pSB1C3 vector and flanked by the same pre- and suffix sequences, we should be able to amplify all SPs via PCR using the BioBrick pre- and suffix as primers. To cope with this issue of having 74 SPs, we decided to break down the 74 SP into Signal Peptide Mixes (SPM), each consisting of maximal 20 SPs (Table 2). We have carefully evaluated the maximal number of SPs within each mix to increase the robustness of the PCR (see the Results section for more details). Followed by the amplification of all SPM subsets, they can be easily purified and applied in a shotgun ligation approach with the protein of interest and the EV.
We demonstrate the applicability of this approach with three different proteins: the α-Amylase of B. subtilis, sfGFP and mCherry. For each protein we applied a protein dependent assay and tested supernatants of positive transformed B. subtilis clones. We correlated protein activities with the secretion efficiency. In a final step, we sequenced strains which showed highest protein activities in order to identify the SPs. Thus, we were able to identify the most potent combination for a distinct SP with the given POI.
Detailed methods can be found in our protocol collection section. All primers used can be found in our primer collection table down below.
Amplification of the Signal Peptide Mixes via optimized multi-template PCR
So far, no direct correlation between the perfect combination of signal peptide and downstream sequence to gain optimal secretion levels is known. Thus, the problem of having to create one clone per combination of SP and protein of interest remains. Therefore, we created the so-called Signal Peptide Mixes (SPMs), a set of libraries with each containing equal concentrations of up to twenty distinct SPs which can be easily enriched via multi-template PCR. The amplified SPs can then be combined with our Signal Peptide Evaluation Vector (SP-EV) and the gene of interest. (For more details see the protocol in the end of the Results section.)
All 74 SPs which we provide were therefore aliquoted to 0.5 ng/μL and assigned to one (a, b, c or d) SPM subsets. The table below gives an overview.
SPM subset c
Cloning Signal Peptides with the Signal Peptide-Evaluation Vector
Following the evaluation of the multi-template PCR amplification of the SPs, we established a Standard Operating Procedure (SOP) protocol for cloning the SPs using the Signal Peptide-Evaluation Vector (SP-EV). This SOP describes the high-throughput approach of screening SPs with a POI. While our project is based in B. subtilis and we use E. coli as cloning host, we believe this SOP can be applied to any organism which is able to perform secretion via the Sec pathway.
The detailed SOP protocols for working with the Evaluation Vector and the Signal Peptide Toolbox can be found down below at the end of the Results section "High throughput screening procedure for B. subtilis". The GIF (Figure 4) above summarizes graphically the steps neccessary to set up your individual SP-EV.
High throughput screening procedure for B. subtilis
After various adjustments to improve the applicability of the Signal Peptide Toolbox, we developed a high throughput screening procedure tailored to fit our model organism B. subtilis and proceeded to identify the most potent SPs for highest secretion of B. subtilis' α-Amylase.
Since we wanted to evaluate amylase secretion efficiency, we performed our transformation into a starch degradation-deficient B. subtilis strain (TMB3547). This strain contains a disruption of the amyE gene, due to the insertion of Pveg-lacZ. Fortunately, the strain still contains the necessary flanking regions for homologous recombination of the pBS1C vector. Thus, positive integration of the pBS1C-SPM-amyE construct lead to white colonies, when plated on X-Gal containing agar plates. (Figure 5, A)
We obtained colonies from each transformation (SPM a, b, c or d with amyE in the EV) and transferred colonies to: a starch containing screening agar plate (to check for vector integration into the B. subtilis genome) and a second backup agar plate which was spiked with chloramphenicol (to maintain each colony). In our setup, we included a negative control (TMB3547, non starch degrading) and a positive control (W168, native amyE and thereby native amylase secretion).
Identification of the most potent Signal Peptides for sfGFP and mCherry
The screening procedure for identifying the best SPs for highest secretion of sfGFP and mCherry were conducted as stated in the detailed SOP protocols for working with the Evaluation Vector and the Signal Peptide Toolbox above.
For sfGFP, we applied a microplate reader based fluorescence assay where we performed an endpoint measurement at wavelengths set to 480 nm for excitation and 510 nm emission and normalized the generated data via the clones' OD600 values to identify the most potent combinations of SPs and sfGFP. For mCherry, we performed a second microplate reader based fluorescence assay, an endpoint measurement at wavelengths set to 585 nm for excitation and 615 nm for emission. The data was normalized over the clones' OD600 values. In a final step, we identified the most potent SP‑sfGFP and SP‑mCherry combinations via sequencing (Figures 8, 9).
Our team developed and proved the applicability of a powerful toolbox to quickly screen via a high throughput procedure for improved secretion of proteins in bacteria - the Signal Peptide Toolbox. We are very much sure that the vision to facilitate Peptidosomes as protein production platform can be achieved. The promising combination of increased protein secretion and physical separation of production host and end-product has endless possible applications.
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