基于类弹性蛋白融合金属硫蛋白的构建及其生物学活性评价

Construction and biological activity of metallothionein fused with ELP

摘要:

金属硫蛋白(metallothionein, MT)在去除重金属、抗氧化和免疫调节等方面发挥重要作用。当前获取天然MT蛋白的首选方法是从组织中提取，工艺复杂且收率很低。近年来涌现多种标签融合后异源表达的设计，如GST或His等。然而后期标签的去除大大降低了收率，因此难以实现工业化生产。类弹性蛋白(elastin-like polypeptides, ELPs)融合技术能够实现目标蛋白的可溶性表达且纯化工艺简便快捷。本研究将ELP与MT蛋白融合，通过ELP融合显著增加了MT的可溶性表达，采用多次可逆相变循环(inverse transition cycling, ITC)处理等纯化工艺高效简便地获得了纯度97%以上的ELP-MT蛋白。获得的ELP-MT蛋白2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐[2,2ʹ-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt, ABTS]自由基清除率IC₅₀为0.77μmol/L，为维生素E衍生物Trolox的53.7倍，同时表现出较强的1,1-二苯基-2-三硝基肼(1,1-diphenyl-2-trinitrohydrazine, DPPH)自由基清除能力。并且ELP-MT对小鼠胚胎成纤维细胞NIH/3T3细胞无增殖毒性，可显著促进NIH/3T3细胞黏附和迁移，具有良好的生物相容性。本研究构建的ELP-MT融合蛋白同时具有金属硫蛋白和弹性蛋白的特性，为重组MT蛋白的规模化生产和其在食品保健及化妆品领域的应用开发奠定了技术基础。

关键词:金属硫蛋白;类弹性蛋白;可溶性表达;多次可逆相变循环纯化;生物学活性

Abstract:

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2ʹ-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.

Key words:metallothionein (MT);elastin-like polypeptides (ELPs);soluble expression;purification by inverse transition cycling (ITC);biological activity