Our approach was to combine the high epitope density offered by the bacterial flagellum as a scaffold with the modular advantages of a Streptavidin-Strep-tag system. We planned to fuse an EpCAM-binding DARPin Ec1 with a tetrameric streptavidin, increasing the local concentration of the construct for targeting EpCAM-positive tumor cells. A Strep-tag was introduced into the D2 domain of Salmonella Flagellin, the combined construct was inserted into Bacillus subtilis Flagellin (Hag). Experiments have shown that mutants with D23 domain express the modified Flagellin and are motile. Therefore, the Strep-tag and D2 domain were inserted into hag with additional GSGS linkers. The gene fragment encoding this design was synthesized by Integrated DNA Technologies.
The Hag-D2-Strep domain was inserted into the linearized pMAD cloning vector by Gibson assembly. B. subtilis was transformed with the resulting plasmid piGEM027. Hag-D2-Strep was additionally cloned into pET24d for overexpression and crystallization.
Test expression of Hag-D2-Strep
After cloning procedures were successfully completed, a test expression of Hag-D2-Strep was performed. Therefore, E. coli BL21(DE3) was transformed with piGEM-030. To prevent the monomeric protein from assembling into polymeric filaments, a vector containing a chaperone (FliS) was co-transformed. For the expression test, a 20 mL culture was grown to an optical density (OD) of 0.7 and induced with 1 µM IPTG. Pre-induction (PI) and induction (I) samples were taken and prepared for SDS-PAGE analysis.
Gel analysis reveals that the transformed E. coli BL21(DE3) co-express the monomeric Hag-D2-Strep together with FliS. Hag-D2-Strep runs at the expected size so that an expression culture could be inoculated over night for autoinduction with lactose.
Overexpression and purification
Since the expression test was successful, protein expression was scaled up. For that purpose, 2 x 1 L LB with the appropriate antibiotic was inoculated with every clone on the co-transformation plate. Protein expression was induced through addition of lactose (12,5 g/L), and the cells were cultured over night (ON) at 30 °C.
Ni-NTA and gel filtration purification of Hag-D2-Strep/FliS complex
Cells were harvested and the pellet was resuspended in buffer A before lysing the cells with the microfluidizer. After centrifugation the clear supernatant (Load) was loaded on a Ni-NTA HisTrap. Flow through (FT), wash (W) and elution (E) were analyzed on an SDS-PAGE.
The calculated molecular mass of Hag-D2-Strep is 47,7 kDa. Gel analysis revealed that a band of the expected size is present in all loaded samples and that most of the protein is present in the elution fraction. The eluted protein was concentrated and further purified by gel filtration. The fractions covering the peak were analyzed on an SDS-PAGE gel. The protein of interest was present in all analyzed fractions. Therefore, the peak fractions were pooled, concentrated and stored at -80 °C until further use.
1. Pull-down with Hag-D2-Strep & Streptactin-Beads
Purified Hag-D2-Strep was employed in a pull-down assay with Streptavidin beads received from Wieland Steinchen. The aim of this pull-down was to see if the Strep-tag in the Flagellin monomers can interact with the Streptavidin beads.
|1||Strep-beads without protein|
|2||Strep-beads + Hag-D2-Strep|
|3||GST-beads + Hag-D2-Strep|
|4||Strep-beads + Hag-D2-Strep|
|5||GST-beads + Hag-D2-Strep|
The pull-down assay shows that Hag-D2-Strep interacts with the Streptactin beads. In the next step, a competitive pull-down should be performed in order to test the interaction between Hag-D2-Strep and StrepDARPidin.
2. Competitive pull down with Hag-D2-Strep & StrepDARPidin
The purified Hag-D2-Strep was employed in a pull-down assay with purified StrepDARPidin. The pull-down should indicate if the Strep-tag in the Flagellin monomers can interact with the StrepDARPidin, especially with the Streptavidin subunit.
|2||Hag-D2-Strep + StrepDARPidin|
|4||Hag-D2-Strep + StrepDARPidin [1:5]|
|5||Hag-D2-Strep + StrepDARPidin [1:1]|
Altough both proteins were stable in the same buffer (1xPBS+2,5% glycerol) adding of Hag-D2-Strep to the StrepDARPidin led to precipitation. Purified Hag was used as a control and did not show precipitation after adding it to StrepDARPidin. The competitive pull-down shows that just a small amount of protein remains in the supernatant. Hag-D2-Strep seems to be partially stable in the soluble phase, although most of the protein precipitates. We hypothesize that the Strep-tag/Streptavidin binding interaction is very strong, perhaps causing misfolding of Flagellin and thus precipitation. We plan to isolate flagella to determine whether the same reaction takes place.
3. Western Blot Hag-D2-Strep and anti-Strep-Tag-AB
In order to prove that the Strep-tag is present in the Flagellin monomers of the Hag-D2-Strep strain, a Western Blot was performed. By using anti-Strep-tag-antibodies, the presence of the Strep-tag was indicated by a band of the expected size, with no observable wild type band.
4. Motility assays
The Hag-D2-Strep B. subtilis strain was generated in order to isolate the filaments as scaffolds for Streptavidin-fused hybrid proteins. Swarming and swimming assays were performed to analyze the motility of this strain. A defined amount of cells was spotted on the middle of these plates and incubated at 37 °C.
B. subtilis motility was observed for 5.5 h. At each time point, the progress of B. subtilis motility was marked with a line. Evaluation of the swimming and swarming assays lead to the conclusion that the B. subtilis strain with the inserted D2-Strep domain is able to swim and swarm, although it is slower than the wild type.
To explore the reason for this reduced motility, B. subtilis wild type and mutant filaments should be compared by electron microscopy.
5. Electron microscopy
Negative staining transmission electron microscopy images from B. subtilis WT3610 and Hag-D2-Strep were prepared.
Electron micrographs of uranyl-acetate stained B. subtilis cells. (A) Whole cell of wild type strain 3610 (B) Whole cell of Flagellin-D2-Strep strain. Scalebar represents 1 μm. (C) Magnification of flagellar filaments from 3610 wild type strain. Scalebar represents 100 nm (D) Magnification of flagellar filaments from Flagellin-D2-Strep strain. Scalebar represents 300 nm.
Evaluation of electron microscopy reveals that B. subtilis Hag-D2-Strep can assemble flagella in a wild type-like manner, although fewer flagella are present than in wild type.
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