title: Site-specific in situ growth of a cyclized protein-polymer conjugate with improved stability and tumor retention.
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.
authors: Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Jin
authors: J
authors: Hu
authors: Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Wenguo
authors: W
authors: Zhao
authors: Protein Chemistry Facility, School of Biological Sciences, Tsinghua University, Beijing 100084, China.
authors: Yan
authors: Y
authors: Gao
authors: Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
authors: Mengmeng
authors: M
authors: Sun
authors: Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors: Yen
authors: Y
authors: Wei
authors: Protein Chemistry Facility, School of Biological Sciences, Tsinghua University, Beijing 100084, China.
authors: Haiteng
authors: H
authors: Deng
authors: Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address: gaoweiping@tsinghua.edu.cn.
authors: Weiping
authors: W
authors: Gao
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001593:Benzyl Compounds / Q000737:chemistry; D001672:Biocompatible Materials / Q000737:chemistry*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011724:Pyridazines / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D001593:Benzyl Compounds; D001672:Biocompatible Materials; D011108:Polymers; D011506:Proteins; D011724:Pyridazines; D011804:Quinolines; C057465:2-(2-dimethylaminoethyl)-5-benzylidene-6-methyl-3-pyridazinone; D049452:Green Fluorescent Proteins; C047117:bicinchoninic acid; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Atom transfer radical polymerization; Protein delivery; Protein engineering; Protein-polymer conjugate