کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021967 1069272 2007 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis
چکیده انگلیسی

The Ca2+-triggered luciferin-binding protein of Renilla reniformis (RLBP) is a non-covalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin). The gene encoding apoRLBP with 552 nucleotides has been synthesized by assembly PCR methods with synthetic oligonucleotides, and the histidine-tagged apoRLBP expressed as a soluble form in the periplasmic space of Escherichia coli cells. The apoRLBP was purified by nickel chelate chromatography and the procedure yielded 18.2 mg of recombinant apoRLBP from 80 ml of cultured cells with purity greater than 95%. The purified apoRLBP was converted to RLBP by incubation with coelenterazine in the presence of dithiothreitol and the purity of recombinant RLBP was estimated to be over 95% by comparison with the absorption spectral data of native RLBP. When RLBP mixed with Ca2+, coelenterazine was dissociated from RLBP and was utilized for the luminescence reaction of Renilla luciferase. Also semi-synthetic RLBPs with h-, e-, and Bis-coelenterazines were prepared and characterized.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 52, Issue 1, March 2007, Pages 66–73
نویسندگان
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