Part:BBa_K4359009

ClyA-3XFLAG-THP

Introduction

ClyA-3XFLAG-THP is the composite part derived from assembling Cytolysin A (BBa_K4359001), (SSSSG)x2 serine glycine linker (BBa_K4359003), 3X FLAG tag (BBa_K4359007) and tumour homing peptide (BBa_K4359006)

Biology

Cytolysin A acts as a localization signal and targets the protein to the bacterial outer membrane. Subsequently, OMVs derived from the outer membrane carry ClyA-3XFLAG-THP. The serine-glycine linker is flexible and joins ClyA with the subsequent domains. The 3X FLAG epitope tag was added to enable antibody-based detection in Western blots and immunofluorescence. 3X FLAG was chosen over 1X FLAG due to its increased sensitivity. The THP targets the receptor CX3CR1. Detailed information on each basic part can be found on their corresponding pages. This part is derived from ClyA-Myc-Affi (BBa_K4359006) by replacing the myc tag with the 3XFLAG tag, and the anti-HER2 affibody with the THP.

This protein is designed to be displayed on the surface of bacterial outer membrane vesicles (OMVs) and target them to breast cancer cells overexpressing HER2. ClyA contains disulphide bonds and requires strains with periplasmic disulphide bond reduction machinery, such as dsba.

Usage

ClyA-3XFLAG-THP was cloned into pGEX-4T1 between the EcoN1 and BamH1 sites. pGEX-4T1 is compatible with expression in K12 strains which lack a genomic copy of the T7 RNA polymerase (other vectors such as pET are incompatible). The expression of ClyA-3XFLAG-THP was under the control of the IPTG-inducible tac operon. We tested the expression with 1mM of IPTG.

SDS PAGE gel indicating the expression of ClyA-3XFLAG-THP in E.coli cell pellets. An overnight culture was diluted 1/100 into 25ml of LB ampicillin and grown at 37C till mid-log. The culture was induced with IPTG (final concentration 1mM) and grown for 18h at 20C. Cells were pelleted by centrifugation and run on SDS PAGE, followed by Coomassie Brilliant Blue staining. A band at 38kDa was visible (red arrow), corresponding to the molecular weight of ClyA-V5-Affi. Lower molecular weight bands (black) are also visible following induction.

We verified that ClyA-3XFLAG-THP was produced following IPTG induction. We isolated THP-OMVs (OMVs expressing ClyA-3XFLAG-THP) and that it is translocated to the OMV fraction.

Anti-FLAG Western blot of bacterial cells (before and after induction) and OMV fraction. Equal volumes were loaded for the induced and uninduced cell samples. A band at 38 kDa corresponding to ClyA-3XFLAG-THP is visible in all lanes, confirming the production and localization of ClyA-3XFLAG-THP to the OMV fraction. A lower weight band (~27 kDa) is visible in the induced cells. From earlier SDS PAGE analysis, we noted low levels of expression occurring prior to induction as well. This possibly due to the leaky natures of the lac operon.

The ability of THP-OMVs expressing ClyA-V5-Affi to selectively internalise in a cancer cell overexpressing the target of the THP, CX3CR1 is crucial. We tested the uptake of THP-OMVs in SK-BR-3, a HER2+ breast cancer cell line. Cells were incubated with 5ug of THP-OMVs (quantity indicated is total protein concentration) for 4-5h at 37C. Cells were washed, fixed, stained and visualised by confocal microscopy.

THP-OMVs were incubated for 4 hours with SK-BR-3 cells. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal is detected from the cytoplasm of the cell, indicating internalisation.

We tracked the internalisation of THP-OMVs expressing ClyA-3XFLAG-THP along with Affi-OMVs expressing ClyA-V5-Affi(BBa_K4359008). Together, Affi-OMVs and THP-OMVs comprise our system Duonco. For this experiment we co-incubated 5ug of Affi-OMVs and 5ug of THP-OMVs with SK-BR-3 cells for 4-5h at 37C. Cells were washed, fixed, stained and visualised by confocal microscopy.

Affi-OMVs and THP-OMVs were co-incubated for 4 hours with SK-BR-3 cells. Affi-OMVs (red) expressing ClyA-V5-Affi are visualised by anti-V5 antibodies and anti-rabbit Alexa Fluor 633. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal from both OMVs was detected from the cytoplasm, suggesting internalisation.

We compared this to internalisation in HEK 293T, a cell line with comparatively low expression of CX3CR1. A similar procedure was followed. We also tested the uptake of Affi-OMVs expressing ClyA-V5-Affi [http://https://parts.igem.org/Part:BBa_K4359008(BBa_K4359008)]. Cells were co-incubated with 5ug of Affi-OMVs and 5ug of THP-OMVs(quantity indicated is total protein concentration) for 4-5h at 37C. Cells were washed, fixed, stained and visualised by confocal microscopy.

Affi-OMVs and THP-OMVs were incubated for 4 hours with HEK 293T cells. Affi-OMVs (red) expressing ClyA-V5-Affi are visualised by anti-V5 antibodies and anti-rabbit Alexa Fluor 633. THP-OMVs (green) expressing ClyA-3XFLAG-THP are visualised by anti-FLAG antibodies and anti-mouse Alexa Fluor 488. Signal was detected from the extracellular media.

We generally observed that in SK-BR-3 cells, THP-OMVs were localised to the cell membrane or cell interior, while for HEK 293, THP-OMVs remained as aggregates in the media and did associate with the cell membranes. This provides encouraging evidence for that ClyA-3XFLAG-THP targets OMVs for selective uptake in cells overexpressing the target marker.

Safety

The safety of ClyA-3XFLAG-THP was evaluated. The MTT assay is a colorimetric assay for assessing cell metabolic activity and can be used to measure cell viability. In this case, we aimed to measure cell viability following treatments of different concentrations of THP-OMVs expressing ClyA-3XFLAG-THP, as compared to a control treatment of PBS. SK-BR-3 cells were utilized for this assay. The mean percentage viability is reported as the viability of THP-treated SKBR3 cells normalized to the mean viability following PBS treatment

Cytotoxicity of THP-OMVs expressing ClyA-3XFLAG-THP as measured using the MTT assay with SK-BR-3 cell cultures. Values are reported as mean ± standard error of mean of three biological replicates.