Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
Protein Chemistry Facility, School of Biological Sciences, Tsinghua University, Beijing 100084, China.
Biomaterials. 2015 Apr;47:13-9. doi: 10.1016/j.biomaterials.2015.01.002. Epub 2015 Feb 2.
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.
治疗性蛋白的一个主要缺点是，在储存、运输和使用过程中，它们对外界应激因素不稳定。在这里，我们报告了一种定点原位生长的环化蛋白-聚合物缀合物，其具有改善的体外和体内稳定性。绿色荧光蛋白（GFP）在其 N 端和 C 端分别与两个天冬酰胺酶识别序列五肽甘氨酸和 LPETG 融合，得到线性 GFP（l-GFP）。l-GFP 通过天冬酰胺酶催化环化生成环化 GFP（c-GFP）。通过选择性地将马来酰亚胺功能化的原子转移自由基聚合（ATRP）引发剂连接到 GFP 的 C 末端遗传工程化的游离半胱氨酸残基上，形成大分子引发剂（c-GFP-Br）。随后，从 c-GFP-Br 原位聚合 OEGMA 生成定点（C 末端）和化学计量（1:1）的 c-GFP-POEGMA 缀合物，转化率几乎为定量，且保留了高活性。值得注意的是，与 l-GFP 及其对应物 l-GFP-POEGMA 相比，c-GFP-POEGMA 缀合物的热稳定性分别提高了 9 倍和 310 倍。此外，与 l-GFP 和 l-GFP-POEGMA 相比，该缀合物在肿瘤中的保留时间显著延长。所开发的方法可能适用于各种治疗性蛋白，以提高其体外和体内稳定性。
