Polymer Biotechnology Group, Microbial and Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas-CSIC, Madrid, Spain.
Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy-Spanish National Research Council (SusPlast-CSIC), Madrid, Spain.
Appl Environ Microbiol. 2020 Jun 2;86(12). doi: 10.1128/AEM.00570-20.
Phasin PhaF from consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both and The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP-β-galactosidase and MinP-CueO from Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both and .
phaF 来自 ，由一个模块化蛋白质组成，其 N 端结构域（BioF）已被证明负责与聚羟基烷酸酯（PHA）颗粒结合。BioF 已被用于生物技术目的，作为亲和标签，用于将融合蛋白功能化到 PHA 珠上，这在 和 中都有报道。该结构域的结构模型表明，它具有一个两亲性的 α-螺旋构象，疏水性残基朝向 PHA 颗粒。在这项工作中，我们分析了天然 BioF 标签的平均疏水性和疏水力矩，以合理设计保持对颗粒亲和力的更短版本。含有绿色荧光蛋白（GFP）融合到 BioF 衍生物的杂合蛋白被研究用于 PHA 上的 定位、在 PHA 颗粒表面对 pH、温度和离子强度的稳定性，以及它们对 PHA 合成的可能影响。基于获得的结果，提出了一个用于 PHA 功能化的最小化 BioF 标签（MinP），它保留了类似的结合特性，但由于其尺寸减小，具有有吸引力的生物技术潜力。通过分析 MinP-β-半乳糖苷酶和 MinP-CueO 融合蛋白的功能和稳定性，进一步验证了 MinP 标签的有效性。聚羟基烷酸酯（PHAs）是生物相容性、无毒和可生物降解的生物聚合物，具有在工业和医学领域的特殊应用。PHA 颗粒的复杂结构可被利用作为一个工具箱，在其表面展示感兴趣的分子。phaSins 是颗粒上最丰富的蛋白质组，被用作锚定标签，以获得用于高亲和力生物分离、酶固定化、诊断或细胞靶向的功能化 PHA 珠。在这里，已经证明一个基于先前设计的 BioF 标签的更短模块能够保持对 PHA 颗粒的亲和力，具有更高的稳定性和类似的功能化效率。使用更短的 67%肽，保持整个蛋白质的结合特性，这对于在 PHA 表面固定重组蛋白是一个优势，这在 和 中都有报道。
