Part:BBa_K4359006

ClyA-Myc-Affi

Introduction

ClyA-Myc-Affi is the composite part derived from assembling Cytolysin A (BBa_K4359000), (SSSSG)x2 serine glycine linker (BBa_K4359003), myc-tag (BBa_K82303) and anti-HER2 affibody (BBa_K4359002). It is used to target the human epidermal growth factor receptor 2 (HER2) protein.

Biology

Cytolysin A acts as a localization signal and targets the protein to the bacterial outer membrane. Subsequently, outer membrane vesicles (OMVs) derived from the outer membrane carry ClyA-Myc-Affi. The serine-glycine linker is flexible and joins ClyA with the C terminus domains. The myc epitope tag was added to enable antibody-based detection in Western blots and immunofluorescence. The anti-HER2 affibody is an affinity protein against HER2. Detailed information on each basic part can be found on their corresponding pages.

This protein is designed to be displayed on the surface of OMVs, and target them to breast cancer cells over expressing HER2. ClyA contains disulphide bonds and requires strains with periplasmic disulphide bond reduction machinery, such as dsba.

Usage

ClyA-Myc-Affi was cloned into pGEX-4T1, between the EcoN1 and BamH1 sites. pGEX-4T1 is compatible for expression in K12 strains which lack a genomic copy of the T7 RNA polymerase (and hence cannot use other expresion vectors such as pET). The expression of ClyA-Myc-Affi was under the control of the IPTG-inducible tac operon. We tested expression with 1mM of IPTG.

SDS PAGE gel indicating the expression of ClyA-Myc-Affi 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 45kDa was visible, corresponding to the molecular weight of ClyA-Myc-Affi

OMVs were isolated from E.coli cultures expressing ClyA-Myc-Affi. ClyA-Myc-Affi correctly localizes to the OMV fraction, as noted from the following SDS PAGE gel.

(A) SDS PAGE gel image indicating expression of ClyA-Myc-Affi (red arrow) in the bacterial cells following IPTG induction. A faint band is also visible prior to induction, in line with the known leaky behaviour of the lac operon. A faint band of the same molecular weight (black arrow) is visible in Affi-OMVs but is absent in native OMVs. Lower molecular weight bands are also visible in both cells post-induction and Affi-OMV(green arrow) but absent in cells pre-induction. (B) SDS PAGE gel image of Affi-OMV sample from (A). Higher amount was loaded to visualise the bands more clearly. ClyA-Myc-Affi is visible at 45kDa (black arrow), along with the lower molecular weight band noted earlier (green arrow).

Anti-myc western blots were used to detect ClyA-Myc-Affi in cell pellets and OMVs.

(A) Western blot showing the presence of ClyA-Myc-Affi in E.coli cell pellets following IPTG induction. The membrane was probed with rabbit anti-myc tag primary antibody. A band was detected at the expected size of 45 kDa. An additional higher molecular weight band was also noted. (B) The presence of ClyA-Myc-Affi was also noted in OMVs. Signal detected was proportional to the amount of OMVs loaded.