非天然氨基酸改造水通道蛋白分子结构提高仿生膜稳定性的研究

Aquaporins are water channel proteins within biological membranes, which have extraordinary water permeability and selectivity. The preparation of biomimic membrane containing aquaporins has been a hot spot for water processing technology. However, the instability of such bionical structure because of weak linkage between AQP and phospholipid bilayers has severely limited its industrial applications. Based on the bioinformatic analysis, and with the post-translation modification of aldehyde group and codon expansion technique, an efficient in vitro and in vivo expression system will be developed to obtain the modified AQP with unnatural amino acids. Then the covalent crosslink between AqpZ and phospholipid bilayers will be realized through the introduced functional group mediated biological orthogonal reaction, and finally biomimic membrane with both high water permeability and stability shall be fabricated. The designed innovative crosslink of aquaporin with lipid proteoliposomes, which was mediated by unnatural amino acids, could significantly improve the stability of AQP containing biomimic membrane. The establishment and application of this technology shall provide useful insights on developing next generation of biomimetic membranes, which could be extensively applied in seawater desalination, wastewater treatment，separation of industrial products and special military areas, etc.

水通道蛋白(AQP) 是一种对水分子具有高选择性和渗透性的跨膜蛋白，基于AQP优异的性能，含AQP仿生膜制备已成为水处理领域的研究热点。但这类仿生膜因AQP与膜磷脂层的结合能力弱而导致的结构不稳定性问题严重限制了其在很多水处理领域的工业应用。本项目结合生物信息学分析，采用密码子拓展和翻译后醛基化修饰，建立两种非天然氨基酸改造水通道蛋白质分子结构的新技术，实现含非天然氨基酸AQP的体内外高效制备。在此基础上，通过非天然氨基酸介导的生物正交反应，实现AQP与磷脂的共价交联，制备高稳定性的仿生水过滤膜。这一工作不仅有助于建立一个非天然氨基酸改造膜蛋白的技术平台，而且首次提出水通道蛋白质与仿生膜材料（磷脂）分子之间共价交联的新思路，有可能革命性地提高含水通道蛋白质仿生膜的稳定性，进而推进这类含AQP仿生膜在海水脱盐、污水处理、工业膜分离、特殊军工航天等多个水处理领域的工业化应用。

本项目采用生物信息学分析和蛋白质翻译后修饰技术，建立了非天然氨基酸改造水通道蛋白质分子结构的新技术，成功将非天然氨基酸pPpa编入水通道蛋白AQPZ和AQPSS9，实现了含非天然氨基酸AQPZ（P-AQPZ）和AQPSS9(P-AQPSS9)的高效表达以及高稳定性仿生膜的制备。. 经优化,在无细胞表达体系中P-AQPZ的表达水平达到48 mg/L，在添加1.0% Brij-78的条件下，P-AQPZ的可溶表达部分经镍柱亲和层析纯化，浓度达到223ug/mL,达到制膜要求。在此基础上，通过非天然氨基酸介导的生物正交反应，实现了AQP与磷脂的共价交联。采用停留光谱测定了嵌入P-AQPZ的脂蛋白体的透水功能，其水通透因子Pf值为3.42×10-4m/s，是AQPZ脂蛋白体（1.23×10-4m/s）的2.8倍，表明非天然的编入使AQPZ与磷脂的亲和性增强，具有更好的滤水活性。采用静电吸附原理和层层自组装方法制备了高稳定性的仿生水过滤膜并对其进行了功能评价。结果表明，无论是水通量还是截盐率，含P-AQPZ的仿生膜（水通量19.9 L·m-2·h-1，截盐率68.3%）相较于含AQPZ的仿生膜（水通量18.7 L·m-2·h-1，截盐率60.7%）均有所提高和改善。对另外一种水通道蛋白AQPSS9的研究，也得到了类似的结果。. 研究表明，这一非天然氨基酸改造水通道蛋白的技术平台，可以提高这类含AQP仿生膜的稳定性，为这一类仿生膜的进一步应用研究奠定了良好基础。