Part:BBa_K1766012:Experience

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••••• BGU Israel 2015

Our use of the part was to express it on the plasma membrane of human cancer cells, which can then be specifically targeted by a compatible drug or toxin (Fig. 1). Fig. 1. Possible applications of Affibody expression in cancer cells. Results 1. Cloning We cloned the Affibody [BBa_K1766012] into our AAV vector for expression under CMV promoter (Fig. 2). A B Fig. 2. A. Cloning map of Affibody into AAV vector. B. Successful cloning results. 2. Affibody expression in human cancer cells Affibody-AAV was produced, and used for transduction of human cancer cell line (HT1080 fibrosarcoma cell line). As the Affibody was predesigned to include 3xFLAG peptide (DYKDDDDK) in its sequence, we used anti-FLAG-specific primary antibody to specifically detect the protein in human cancer cells (HT1080 fibrosarcoma) using immunofluorescence. We used two staining protocols, with and w/o permeabilization step, in an attempt to distinguish between extracellular membrane localization and intracellular expression. As shown in Fig. 3, transfection with Affibody-expressing vector resulted in detectable Affibody expression only when permeabilization step was applied. This could indicate the the protein construct is expressed, but it is not correctly transported and localized to the outer membrane, or FLAG sequence (the antigen for the primary Ab) is located inside the cytoplasm. Fig. 3. Affibody expression in human cancer cells. A-B. Representative images of Affibody expression in human fibrosarcoma cell line HT1080, using Alexa-488-conjugated secondary antibody (methanol fixation & permeabilization). C. Lack of staining w/o permeabilization step (fixation with 4% PFA in PBS). D. Negative control (w/o primary antibody). Conclusion and future aspects Affibody construct could be successfully expressed in human cancer cells. However, the design of the construct should be optimized to ensure the expression on the outer cell membrane for its subsequent use for cancer cell tagging and targeting. For detection, primary Ab against Affibody part of the construct is preferable, in order to clearly validate the expression on the outer membrane. For more information visit here.

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