Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.
Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.
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DOI：
10.1021/acs.biomac.5b00549
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发表时间：
2015-08-10
影响因子：
6.2
通讯作者：
Griffith LG
中科院分区：
文献类型：
--
作者：
Cambria E;Renggli K;Ahrens CC;Cook CD;Kroll C;Krueger AT;Imperiali B;Griffith LG
合成的细胞外基质广泛用于再生医学和作为构建体外生理培养模型的工具。合成水凝胶显示出有利的物理性质，但用大肽或蛋白质修饰具有挑战性。在这里，提出了一种简便、温和的酶促后接枝方法。分选酶介导的连接用于将融合至GGG连接基序（GGG-EGF）的人表皮生长因子缀合至含有分选酶LPRTG底物的聚（乙二醇）（PEG）水凝胶。然后利用分选酶反应的可逆性从水凝胶中切割束缚的EGF用于分析。反应上清液和连接后水凝胶的分析表明，栓系的EGF的量随着水凝胶中LPRTG或上清液中GGG-EGF的增加而增加。分选酶系的EGF具有生物活性，如通过刺激原代人肝细胞和子宫内膜上皮细胞中的DNA合成所证明的。分选酶介导的连接和切割反应的简单性、特异性和可逆性使其成为水凝胶改性的有吸引力的方法。
Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.