Lovrinovic Marina, Niemeyer Christof M
Universität Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund, Germany.
Biochem Biophys Res Commun. 2005 Sep 30;335(3):943-8. doi: 10.1016/j.bbrc.2005.08.001.
We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.
我们报道了一种用半胱氨酸部分对市售氨基修饰的DNA寡核苷酸进行共价修饰的快速方法。所得的DNA-半胱氨酸缀合物是通过表达蛋白连接（EPL）高效制备共价DNA-蛋白质缀合物的通用试剂。EPL方法允许半胱氨酸修饰的DNA寡聚物与重组内含肽融合蛋白进行位点特异性偶联，后者含有一个C端硫酯，能够与N端半胱氨酸化合物进行温和且高度特异性的反应。我们通过一步反应制备了一种半胱氨酸修饰试剂，该试剂能够快速且近乎定量地合成半胱氨酸-DNA缀合物。后者与作为内含肽融合蛋白重组表达的绿色荧光蛋白突变体EYFP进行连接，从而能够以约60%的高产率温和且选择性地形成EYFP-DNA缀合物。我们预计我们的方法有许多应用，从蛋白质微阵列到新兴的纳米生物技术领域。
