Site-specific in situ growth of a cyclized protein-polymer conjugate with improved stability and tumor retention |
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| Affiliation: | 1. Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China;2. Protein Chemistry Facility, School of Biological Sciences, Tsinghua University, Beijing 100084, China;3. Department of Chemistry, Tsinghua University, Beijing 100084, China;1. Department of Pharmaceutical Science, Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada;2. Physiology & Experimental Medicine Research Program, The Hospital for Sick Children, Toronto, Ontario M5T 3H7, Canada;3. Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Hesse 65926, Germany;4. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada;1. Department of Orthodontics, Showa University School of Dentistry, 2-1-1, Kitasenzoku, Ohta-ku, Tokyo 145-8515, Japan;2. Department of Conservative Dentistry, Division of Biomaterials and Engineering, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;3. Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;4. Department of Biochemistry, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;1. Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan, Hubei 430072, PR China;2. Wuhan University Suzhou Research Institute, Suzhou, Jiangsu 215123, PR China |
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| Abstract: | A major disadvantage of therapeutic proteins is their instability to external stressors during storage, transport and use. Here, we report site-specific in situ growth of a cyclized protein-polymer conjugate with improved in vitro and in vivo stability. Green fluorescence protein (GFP) was genetically fused at its N- and C-termini with two sortase recognition sequences pentaglycine and LPETG, respectively to yield a linear GFP (l-GFP). A cyclized GFP (c-GFP) was generated from the l-GFP by sortase-catalyzed cyclization. A maleimide-functionalized atom transfer radical polymerization (ATRP) initiator was selectively attached to a free cysteine residue genetically engineered at the C-terminus of GFP to form a macroinitiator (c-GFP-Br). Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) from the c-GFP-Br generated a site-specific (C-terminal) and stoichiometric (1:1) c-GFP-POEGMA conjugate with almost quantitative conversion and highly retained activity. Notably, the c-GFP-POEGMA conjugate showed 9- and 310-fold increases in thermal stability as compared to the l-GFP and its counterpart l-GFP-POEGMA, respectively. Additionally, the conjugate displayed significantly improved tumor retention relative to the l-GFP and l-GFP-POEGMA. The method developed may be applicable to a variety of therapeutic proteins to improve their in vitro and in vivo stability. |
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| Keywords: | Protein-polymer conjugate Protein engineering Protein delivery Atom transfer radical polymerization |
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