Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

被引:23
作者
Lovrinovic, M [1 ]
Niemeyer, CM [1 ]
机构
[1] Univ Dortmund, Fachbereich Chem Biol Chem Mikrostrukturtechn, D-44227 Dortmund, Germany
关键词
DNA; expressed protein ligation; immobilization; microarrays; protein modification;
D O I
10.1016/j.bbrc.2005.08.001
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2005 Published by Elsevier Inc.
引用
收藏
页码:943 / 948
页数:6
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