Ramirez Miguel A, Chen Zhilei
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station.
J Vis Exp. 2014 Jan 27(83):e51202. doi: 10.3791/51202.
We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 °C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a "docking station peptide" into the hydrogel building block enables convenient incorporation of "docking protein"-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.
我们展示了一种由分裂内含肽催化的蛋白质反式剪接反应介导的高度稳定的蛋白质水凝胶的合成。这种水凝胶的构建模块是两种蛋白质嵌段共聚物，每种都包含作为交联剂的三聚体蛋白质的一个亚基和一个分裂内含肽的一半。一种高度亲水的无规卷曲被插入到其中一种嵌段共聚物中以保持水分。两种蛋白质嵌段共聚物的混合引发内含肽反式剪接反应，产生一个在两端带有交联剂的多肽单元，该单元迅速自组装成水凝胶。这种水凝胶在酸性和碱性条件下、高达50°C的温度以及有机溶剂中都非常稳定。剪切诱导破裂后，水凝胶能迅速重新形成。将“对接站肽”掺入水凝胶构建模块中，能够方便地掺入带有“对接蛋白”标签的目标蛋白。这种水凝胶与组织培养生长培养基兼容，支持20 kDa分子的扩散，并能固定生物活性球状蛋白。通过包封辣根过氧化物酶并证实其活性，证明了内含肽介导的蛋白质水凝胶作为有机溶剂兼容生物催化剂的应用。
