کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1174941 961779 2008 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Escherichia coli biotin protein ligase: characterization and development of a high-throughput assay
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Escherichia coli biotin protein ligase: characterization and development of a high-throughput assay
چکیده انگلیسی

The rapid rise in pathogenic bacteria resistant to current treatments, coupled with the paucity of new therapeutic agents in the pipeline, has resulted in a significant need for new antibiotics. One strategy to overcome resistance requires new chemical entities that inhibit key enzymes in essential metabolic processes that have not been previously targeted and for which there is no preexisting drug resistance. Biotin protein ligase (BPL), required to complete acetyl CoA carboxylase’s capability for fatty acid biosynthesis, is one target that has not yet been fully explored. However, its application in large-scale compound screens has been limited due to the lack of a truly high-throughput assay for enzyme activity. Here we report a novel assay system for BPL from Escherichia coli (BirA). This assay employs fluorescence polarization technology together with a unique peptide substrate for BirA. Additionally, the multiple handling steps and requirement for radiolabeled ligands associated with previous assays have been eliminated. Kinetic analysis of MgATP (Km 0.25 ± 0.01 mM) and biotin (Km 1.45 ± 0.15 μM) binding produced results consistent with published data. Inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, further validated the assay. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, Z′ factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 376, Issue 1, 1 May 2008, Pages 131–136
نویسندگان
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