Urquhart Taylor, Daub Elisabeth, Honek John Frank
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo , 200 University Avenue, Waterloo, Ontario, Canada N2L 3G1.
Bioconjug Chem. 2016 Oct 19;27(10):2276-2280. doi: 10.1021/acs.bioconjchem.6b00460. Epub 2016 Sep 16.
With a mass of ∼1.6 × 10 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, l-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.
噬菌体M13的质量约为1.6×10道尔顿，由大约2700种蛋白质组成，已被用作生物纳米材料制造中的分子支架。为了扩展M13在该领域的多功能性，采用了位点特异性非天然氨基酸掺入法，成功地在该噬菌体pVIII主要衣壳蛋白的特定溶剂暴露位置展示了叠氮官能团。利用编码pVIII蛋白的基因的工程突变体、甲硫氨酸（Met）类似物l-叠氮高丙氨酸（Aha）以及用于噬菌体生产的合适的大肠杆菌Met营养缺陷型，开发了相关条件，以生产标记有超过350个活性叠氮基的M13噬菌体（通过利用应变促进的叠氮-炔环加成的荧光染料标记估计），并且通过透射电子显微镜观察到其能够将叠氮基选择性地附着到5纳米的金纳米颗粒上。还验证了该系统利用化学酰化和生物正交环加成反应进行双重标记的能力。上述策略在基于M13构建块制备更大、更复杂分子结构的组装体时应会特别有利。
