Center for Single Molecule Biophysics, Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York 14214, USA.
Protein Sci. 2011 Jun;20(6):1005-20. doi: 10.1002/pro.633. Epub 2011 Apr 27.
The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.
大肠杆菌单链 DNA 结合蛋白（SSB）是 DNA 代谢中的核心因子，它可以组织基因组维护复合物，并稳定在 DNA 处理过程中产生的单链 DNA（ssDNA）中间体。由于 SSB 的重要性以及为了便于实时研究，我们开发了一种双质粒表达系统来产生新型嵌合 SSB 蛋白。这些嵌合体包含组氨酸标记和荧光蛋白（FP）融合亚基的混合物，可轻松以毫克级别的量纯化，并可在无需进一步修饰的情况下使用，这是对以前生产荧光 SSB 的方法的重大改进。嵌合体在所有测定中保留了野生型的功能，表明 FP 对 SSB 功能没有影响。我们在单分子研究中展示了这些嵌合体的强大功能和实用性，为 RecBCD 的生化机制提供了深入的了解。我们还利用嵌合体首次表明 RecG 和 SSB 在体内相互作用。因此，我们预计本文所述的嵌合体将促进体内、体外和单 DNA 分子研究，这些研究使用的蛋白质在使用前无需进一步修饰。
