蛋白质内含子介导蛛丝功能化平台的建立

Construction of spider silk functional platform via intein trans-splicing

摘要:

为建立高效快捷的蛛丝功能化修饰平台，蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp DnaB的反式剪接作用，在蛋白质水平上将12 kDa泛素相关修饰蛋白 (SUMO) 与蛛丝蛋白 (W2CT) 连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维，但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维 (W) 相似，SUMOW2CT丝纤维 (UW) 不具有超收缩能力和对2%SDS不耐受，但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构，可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能，但修饰上的功能化蛋白仍保持着生物活性，表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立，也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。

Abstract:

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.