Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain.
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CIB-CSIC), Madrid, Spain.
Appl Environ Microbiol. 2018 Jan 31;84(4). doi: 10.1128/AEM.02595-17. Print 2018 Feb 15.
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-β-galactosidase, containing the choline-binding module C-LytA and the β-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-β-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials. Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.
聚羟基脂肪酸酯（PHAs）是一种可生物降解的聚酯，在某些细菌的细胞质中积累。一种有前途的生物技术应用是将这些生物聚合物用作蛋白质固定化的载体。在这里，研究了 KT2440 PhaF 相蛋白（BioF 多肽）的 PHA 结合域作为亲和标签，用于用重组蛋白功能化聚-3-羟基丁酸酯（PHB）颗粒，即全长 PhaF 和两个融合蛋白标记为 BioF（BioF-C-LytA 和 BioF-β-半乳糖苷酶，分别含有胆碱结合模块 C-LytA 和β-半乳糖苷酶酶）。在广泛的 pH 和温度范围内，蛋白质-生物聚酯的相互作用很强且稳定，结合的蛋白质高度免受自降解的影响，而结合强度可以通过涂覆两亲化合物来调节。最后，当在 minibioreactor 中固定化时，BioF-β-半乳糖苷酶在连续几个活性加洗涤循环后仍表现出非常稳定的酶活性。我们的结果表明 PHA 和 BioF 标签具有构建新型生物活性材料的潜力。我们的结果证实了 BioF 亲和标签作为一种多功能工具用于用重组蛋白功能化 PHA 载体的生物技术潜力，从而产生新型生物活性材料。BioF 标签的广泛底物范围可能使几乎所有天然 PHAs 以及具有新型物理化学性质的混合物、共聚物或人工化学修饰衍生物的蛋白质固定化成为可能。此外，通过改变载体的涂层，可以轻松调节蛋白质吸附的强度，为需要严格控制蛋白质负载的生物活性材料的工程设计提供了新的视角。
