Microtiter plate-based screening for the optimization of DNA-protein conjugate synthesis by means of expressed protein ligation

被引:28
作者
Lovrinovic, Marina [1 ]
Niemeyer, Christof M. [1 ]
机构
[1] Univ Dortmund, Fachbereich Chem, D-44227 Dortmund, Germany
关键词
DNA; expressed protein ligation; immobilization; microarrays; protein modifications;
D O I
10.1002/cbic.200600303
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 microtiter plate screening assay for the optimization of the synthesis 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 containing a C-terminal thioester that enables a mild and highly specific reaction with N-terminal cysteine compounds. To screen for optimal reaction conditions, we developed a microtiter plate-based assay that utilizes DNA-directed immobilization of the products formed in the ligation reaction of cysteine-modified DNA oligonucleotides with the model protein thioester of the maltose-binding protein (MBP), recombinantly expressed as an intein-fusion protein in E. coli. The screening assay allowed the rapid quantitative monitoring of various reaction parameters, such as the ratio of the reactants, reaction times, pH and ion strength of the buffer, the influence of various thiol additives and the nature of the chemical linker within the cysteine-bearing DNA oligonucleotide. As the consequence of the assay-based optimization, the ligation of MBP with the oligonucleotide was improved to near quantitative yields.
引用
收藏
页码:61 / 67
页数:7
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