Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia.
Jožef Stefan Institute, Condensed Matter Physics Department, 1000 Ljubljana, Slovenia.
Nucleic Acids Res. 2022 Oct 28;50(19):e113. doi: 10.1093/nar/gkac714.
Encapsulation of a selected DNA molecule in a cell has important implications for bionanotechnology. Non-viral proteins that can be used as nucleic acid containers include proteinaceous subcellular bacterial microcompartments (MCPs) that self-assemble into a selectively permeable protein shell containing an enzymatic core. Here, we adapted a propanediol utilization (Pdu) MCP into a synthetic protein cage to package a specified DNA segment in vivo, thereby enabling subsequent affinity purification. To this end, we engineered the LacI transcription repressor to be routed, together with target DNA, into the lumen of a Strep-tagged Pdu shell. Sequencing of extracted DNA from the affinity-isolated MCPs shows that our strategy results in packaging of a DNA segment carrying multiple LacI binding sites, but not the flanking regions. Furthermore, we used LacI to drive the encapsulation of a DNA segment containing operators for LacI and for a second transcription factor.
将选定的 DNA 分子封装在细胞中对生物纳米技术具有重要意义。可作为核酸容器的非病毒蛋白包括蛋白质亚细胞细菌微隔间（MCP），它们自组装成具有酶核心的选择性渗透蛋白壳。在这里，我们将丙二醇利用（Pdu）MCP 改编为合成蛋白笼，以在体内包装指定的 DNA 片段，从而能够进行随后的亲和纯化。为此，我们设计了 LacI 转录阻遏物，使其与目标 DNA 一起进入 Strep 标记的 Pdu 壳的内腔。从亲和分离的 MCP 中提取的 DNA 测序表明，我们的策略导致包装了带有多个 LacI 结合位点的 DNA 片段，但不包括侧翼区域。此外，我们使用 LacI 驱动包含 LacI 和第二个转录因子的操作子的 DNA 片段的封装。
