RRC ID 73953
Author Inouye S.
Title Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis.
Journal Protein Expr Purif
Abstract 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 18.2mg 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.
Volume 52(1)
Pages 66-73
Published 2007-3-1
DOI 10.1016/j.pep.2006.07.028
PII S1046-5928(06)00246-4
PMID 16997571
MeSH Aequorin / chemistry Aequorin / genetics Amino Acid Sequence Animals Apoenzymes / genetics Apoenzymes / metabolism Apoproteins / metabolism Base Sequence Calcium / pharmacology* Calcium-Binding Proteins / genetics Calcium-Binding Proteins / metabolism* Conserved Sequence Genetic Vectors Luciferases, Renilla / genetics Luciferases, Renilla / metabolism* Molecular Sequence Data Recombinant Proteins / metabolism Renilla / genetics* Renilla / metabolism* Restriction Mapping Sequence Alignment Sequence Homology, Amino Acid
IF 1.513
DNA material pJN-RLBP (RDB19832)