DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker
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ORIGINAL ARTICLE
DARPin Ec1‑LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker Nikta Babaee1,2 · Yeganeh Talebkhan Garoosi1 · Morteza Karimipoor2 · Fatemeh Davami1 · Elham Bayat1 · Hossein Safarpour3 · Fereidoun Mahboudi1 · Farzaneh Barkhordari1 Received: 7 March 2020 / Accepted: 28 August 2020 © Springer Nature B.V. 2020
Abstract This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6 kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells. Keywords DARPin Ec1-LMWP · EpCAM · siRNA · CSC
Introduction
Yeganeh Talebkhan Garoosi and Morteza Karimipoor are cocorrespondence authors Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11033-020-05752-5) contains supplementary material, which is available to authorized users. * Yeganeh Talebkhan Garoosi [email protected]; [email protected] * Morteza Karimipoor [email protected] 1
Biotechnology Research Center, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
2
Biotechnology Research Center, Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran
3
Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran
Cancer is one of the most common multifactorial diseases which cause high rates of mortality and morbidity. It has been well documented that targeted therapy can be more efficient in cancer treatment and in comparison, to the chemotherapy shows less damages to the healthy tissues [1]. One of the strategies for targeted therapy of cancer is the use of monoclonal antibodies or proteins that can specifically bind to the surface tumor markers [2]. Protein scaffolds are the kind
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