Difference between revisions of "Part:BBa K1329000"

Revision as of 18:29, 15 October 2014

StrepDARPidin

Idea

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.

Experiments from Florian Altegoer have shown that mutants with Salmonella D23-domain integrated into Hag (flagellin) express the modified flagellin and are motile. Therefore, the Strep-tag was inserted into Hag with D2 as a linker domain and additional GSGS linkers. The gene fragment encoding this design was synthesized by Integrated DNA Technologies.

Cloning procedure

Cloning started with PCR amplification of the synthesized StrepDARPidin and subsequent restriction and ligation into pET16b. Competent E. coli XL1-Blue and E. coli BL21(DE3) cells were transformed afterwards with the resulting vector for overexpression.

Expression test

Before performing assays with purified StrepDARPidin, protein expression was tested in E. coli BL21(DE3) cells. Pre-induction (PI) and induction (I) samples were collected for SDS-PAGE gel analysis.

Gel analysis reveals positive overexpression of a protein of ca. 30 kDa, the calculated molecular mass of StrepDARPidin.

Overexpression and purification of StrepDARPidin

For the expression of tetrameric StrepDARPidin, E. coli BL21(DE3) cultures containing piGEM-028 (pET16b StrepDARPidin) were induced with lactose and grown overnight at 30 °C.

For the purification of StrepDARPidin, cells were harvested and lysed via a microfluidizer. Initial purification analysis reveals that StrepDARPidin accumulates in inclusion bodies independent of the induction method or concentration of the inducing molecule.

A protocol for the purification of StrepDARPidin from inclusion bodies was developed and optimized (see Notebook). The refolded protein could afterwards be purified by Ni-NTA affinity and gel filtration.

After optimizing the refolding protocol, the refolded load (L) was purified on a 1-mL HisTrap column and, along with the flow-through (FT), wash (W), and elution (E), analyzed on an SDS-PAGE gel. The gel reveals that the monomeric and tetrameric versions of StrepDARPidin are present in the load, flow-through and elution fraction.

Gel filtration

After the Ni-NTA purification, the eluted protein was concentrated for gel filtration. All fractions covering the peak in the chromatogram were analyzed on an SDS-PAGE gel in an uncooked and cooked (heat-denatured) state. Gel analysis reveals that the peak contains StrepDARPidin as a tetramer that can be dissolved by incubation at 95 °C for 10 minutes. The fractions were pooled, concentrated and stored at -80 °C until further use.

Interaction assays

1. Competitive pull-down with Hag-D2-Strep & StrepDARPidin

The purified Hag-D2-Strep assayed in a pull-down 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.

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.

Cancer detection

1. Fluorescence measurements after StrepDARPidin targeting

In order to analyze the binding of purified StrepDARPidin to EpCAM-positive cells, an ELISA-like assay was performed. Caco-2 cells (colon cancer cell line) and A549 cells (lung cancer cell line) were grown in a black 96-well ELISA plate overnight. The cells were incubated with variable concentrations of StrepDARPidin and washed to remove excess protein. Afterwards, cells with bound protein were incubated with anti-His-antibody-Alexa488 conjugate (Qiagen) to target the StrepDARPidins His-tag. After washing away excess antibodies, fluorescence was measured by exciting the cells at 480 nm and measuring the signal at 540 nm.

The measurements show that, in the presence of 25 µM StrepDARPidin, the fluorescence signals are ca. 1,7-1,9-fold higher than in the EpCAM-negative 3T3WT fibroblasts (negative control). The fluorescence signal decreases with decreasing concentration of StrepDARPidin. To confirm the binding of StrepDARPidin to the membrane-associated EpCAM, immunofluorescence microscopy will be performed.

2. Immunofluorescence staining of Caco-2 and A549 cells

In order to confirm the binding of StrepDARPidin to the membrane-associated EpCAM, immunofluorescence staining was performed. Caco-2 cells, A549 cells and fibroblasts were grown on gelatin-coated coverslips and incubated with 25 µM StrepDARPidin. After fixation with formaldehyde, the cells were permeabilized with Triton X-100. The membrane-associated f-actin was stained with phalloidin and the nucleus was stained with DAPI. The fibroblasts and cells without StrepDARPidin incubation served as negative controls.

Microscopy after incubation of EpCAM-positive tumor cells with StrepDARPidin indicates a localization of the protein at the cell membrane. The cell membrane of 3T3WT fibroblasts remains unstained.

Caco-2 cells show a higher intensity of fluorescence after targeting with anti-His-Alexa488 conjugate than A549 which confirms the results of the previously performed ELISA-like assays.

Sequence and Features