Difference between revisions of "Part:BBa K1329000"

Latest revision as of 00:15, 18 October 2014

StrepDARPidin

Idea

Our approach was to combine the scaffold of a tetrameric Streptavidin (Strep) to increase the specificity of DARPin Ec1 for detection of a prominent tumor surface marker of many epithelial-derived cancer cells (like lung & colon cancer cells), called EpCAM (Epithelial Cell Adhesion Molecule) (Went et al. 2006). EpCAM is expressed by every epithelial cell on the basolateral side in a specific ratio while being overexpressed on the surface irregularly by transformed tumor cells. We planned to fuse an EpCAM-binding DARPin Ec1 (Stefan et al. 2011) with a tetrameric Streptavidin increasing the local concentration of the construct for targeting EpCAM-positive tumor cells. In the next step, we wanted to combine the high density of epitopes offered by the filament of the bacterial flagellum as a scaffold with the modular advantages of a Streptavidin-Strep-Tag-system. The sequence for the Streptavidin subunits was taken from Part: BBa_J36848 - Team iGEM2006_Harvard.

The sequence was designed with an N-terminal DARPin Ec1 with linker peptides in between. The StrepDARPidin construct was designed codon-optimized for E. coli commercially by IDT (Integrated DNA Technologies).

Cloning procedure

Template DNA for the StrepDARPidin construct was commercially synthesized by IDT codon-optimized for E. coli and used for PCR amplification. The construct was cloned into a pET16b-expression vector for overexpression of the protein in E. coli BL21 (DE3).

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 revealed positive overexpression of the protein StrepDARPidin with a calculated molecular mass of 31 kDa.

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 over night at 30 °C.

For the purification of StrepDARPidin, cells were harvested and lysed with 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 was 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 revealed 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 and refolding was verified by incubating the eluate for 10 min at 95°C. Gel analysis revealed that the peak contained StrepDARPidin as a tetramer that could be dissolved by incubation at 95 °C for 10 minutes. The fractions were pooled and concentrated.

Interaction assays

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

The purified Hag-D2-Strep was assayed in a pull-down with purified StrepDARPidin. The pull-down should indicate if the Strep-Tag in the Flagellin monomers was able to interact with the StrepDARPidin, especially with the Streptavidin subunit.

1 Hag-D2-Strep, 2 Hag-D2-Strep + StrepDARPidin, 3 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 showed that just a small amount of protein remained in the supernatant. Hag-D2-Strep seemed to be partially stable in the soluble phase, although most of the protein precipitated. We hypothesize that the Strep-tag/Streptavidin binding interaction was 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 over night. 3T3 wild type (WT) fibroblasts (EpCAM negative) were treated the same and used as negative control. 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 StrepDARPidin's His-tag. After washing away remaining antibodies, fluorescence was measured by exciting the cells at 485 nm and measuring the signal at 520 nm.

The measurements showed that in presence of 25 µM StrepDARPidin, the fluorescence signal were about 1.7-1.9-fold higher than in the EpCAM negative 3T3 WT fibroblasts (negative control). The fluorescence signal decreased with decreasing concentration of StrepDARPidin. To confirm the binding of StrepDARPidin to the membrane-associated EpCAM, immunofluorescence microscopy was 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, cells were permeabilized with Triton X-100. The membrane-associated F-Actin was stained with phalloidin-rhodamin and the nucleus was stained with DAPI. The fibroblasts and cells without StrepDARPidin incubation served as negative controls.

Immunofluorescent staining of Caco-2 and 3T3 cells incubated with StrepDARPidin protein. Scale bar represents 5 µm.

Fluorescence intensities of Caco-2 and 3T3 cells in comparison

Immunofluorescent staining of StrepDARPidin treated and untreated A549 cells. Scale bar represents 5 µm.

Microscopy of EpCAM-positive tumor cells incubated with StrepDARPidin indicated a localization of the protein at the cell membrane. The cell membrane of 3T3 WT fibroblasts remained unstained.

Caco-2 cells showed a higher fluorescence intensity after incubating with anti-His-Alexa488 conjugate compared to A549 cells, confirming the results of the previously performed ELISA-like assay.

Sequence and Features