将多巴直接有效地掺入类弹性蛋白中，可以交联成可调谐水凝胶,Biomacromolecules

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将多巴直接有效地掺入类弹性蛋白中，可以交联成可调谐水凝胶
Biomacromolecules ( IF 5.5 ) Pub Date : 2023-03-23 , DOI: 10.1021/acs.biomac.2c01516
Ya-Jiao Zhu ₁ , Sheng-Chen Huang ₁ , Zhi-Gang Qian ₁ , Xiao-Xia Xia ₁

Affiliation

3,4-二羟基苯丙氨酸 (DOPA) 是一种天然存在但非规范的氨基酸，赋予蛋白质聚合物多种化学反应性和新颖的功能。尽管已经做出许多努力将 DOPA 掺入蛋白质中，但掺入效率和生产效价仍然很低，严重阻碍了对这些特殊蛋白质进行生物材料制造的探索。在这里，我们报告了一种有效的生物合成策略来生产大量掺入多巴的结构蛋白，用于制造具有可调机械性能的水凝胶。首先，构建合成基因，编码具有不同比例的酪氨酸残基和分子量的重复性弹性蛋白样蛋白 (RLP) ( M _w). 通过在重组大肠杆菌细胞中内源性酪氨酰-tRNA 合成酶 (TyrRS) 对多巴进行错误氨酰化，将这些基因解码为掺入多巴的RLP。通过开发化学计量学指导的两相培养策略，我们实现了对细菌生长和蛋白质合成阶段的独立控制。这使得掺入 DOPA 的 RLP 能够以克/升的水平超量生产，并且 DOPA 掺入率高达 76-85%。^{然后，通过简单的 DOPA–Fe 3+}络合，纯化的含有 DOPA 的 RLP 成功交联成块状水凝胶。有趣的是，这些水凝胶表现出高度依赖于儿茶酚含量和Mw 的_{粘弹性和自修复特性}RLP的。最后，对分子交联机制的探索表明，蛋白质中较高的多巴含量会导致金属配位和氧化共价交联的同时发生。总之，我们的结果提出了一个有用的平台来生成 DOPA 功能化的蛋白质材料，并提供对基于 DOPA 化学的凝胶化系统的更深入的了解。

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Direct and Efficient Incorporation of DOPA into Resilin-Like Proteins Enables Cross-Linking into Tunable Hydrogels

3,4-Dihydroxyphenylalanine (DOPA), a naturally occurring yet noncanonical amino acid, endows protein polymers with diverse chemical reactivities and novel functionalities. Although many efforts have been made to incorporate DOPA into proteins, the incorporation efficiency and production titer remain low and severely hinder the exploration of these peculiar proteins for biomaterial fabrication. Here, we report an efficient biosynthetic strategy to produce large amounts of DOPA-incorporated structural proteins for the fabrication of hydrogels with tunable mechanical properties. First, synthetic genes were constructed that encode repetitive resilin-like proteins (RLPs) with varying proportions of tyrosine residues and molecular weights (M_w). Decoding of these genes into RLPs incorporated with DOPA was achieved via mis-aminoacylation of DOPA by endogenous tyrosyl-tRNA synthetase (TyrRS) in recombinant Escherichia coli cells. By developing a stoichiometry-guided two-phase culture strategy, we achieved independent control of the bacterial growth and protein synthesis phases. This enabled hyperproduction of the DOPA-incorporated RLPs at gram-per-liter levels and with a high DOPA incorporation yield of 76–85%. The purified DOPA-containing RLPs were then successfully cross-linked into bulk hydrogels via facile DOPA–Fe³⁺ complexations. Interestingly, these hydrogels exhibited viscoelastic and self-healing properties that are highly dependent on the catechol content and M_w of the RLPs. Finally, exploration of the molecular cross-linking mechanisms revealed that higher DOPA contents of the proteins would result in the concomitant occurrence of metal coordination and oxidative covalent cross-linking. In summary, our results suggest a useful platform to generate DOPA-functionalized protein materials and provide deeper insights into the gelation systems based on DOPA chemistry.

更新日期：2023-03-23

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