Barbosa Mariana, Simões Hélvio, Prazeres Duarte Miguel F
iBB-Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Associate Laboratory i4HB-Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Materials (Basel). 2021 Jun 9;14(12):3175. doi: 10.3390/ma14123175.
Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.
通过用一系列生物分子修饰纤维素，可以获得具有新颖和增强功能的材料。这种功能化可以提供具有增强的物理和化学性质以及与特定应用相匹配的生物相互作用控制的定制纤维素基材料。此类生物材料成功的基础之一是有效地控制将相关生物分子以实现所需功能的方式结合到纤维素材料中的能力。在此背景下，我们的主要目标是开发双功能生物分子构建体，用于用感兴趣的生物活性分子精确修饰纤维素水凝胶。主要思路是利用生物分子工程技术来生成和纯化碳水化合物结合模块（CBM）与重要生物实体的不同重组融合体。具体而言，基于CBM的融合体被设计用于实现蛋白质或寡核苷酸与纤维素水凝胶的桥接。这项工作聚焦于将源自 （CBM3） 的纤维素体支架蛋白A的3族CBM与以下物质结合的构建体：（i）N端绿色荧光蛋白（GFP）结构域（GFP-CBM3）；（ii）识别IgG抗体的双Z结构域；以及（iii）C端半胱氨酸（CBM3C）。通过下拉试验评估了CBM融合体将其对应物结合和/或锚定到纤维素水凝胶表面的能力。首先通过荧光显微镜定性地证明了纤维素对GFP-CBM3的捕获。成功证明了融合蛋白的结合、抗体的捕获（通过ZZ-CBM3）以及寡核苷酸的接枝（到CBM3C）。这里描述的生物活性纤维素平台能够将不同的生物分子精确地锚定到纤维素水凝胶上，并且可以对先进的医学诊断传感器或特殊生物材料等的开发做出重大贡献。
