1.13.12.6 additional information biotynilation of the enzyme by attachment of an Avi-tag of a 16-residue peptide to the C-terminus of the luciferase, conjugation to the indocyanine derivative HiLyte Fluor 647 hydrazine via the glycol-chains of the enzyme 700998 1.13.12.6 additional information concept of tumor monitoring using dual luciferases, construction of the expression vector, and evaluation of tumor monitoring systems using dual luciferases from Cypridina noctiluca and firefly Photinus pyralis, overview. The enzymes are expressed in human breast cancercells MDA-MB-231, followed by inoculatin of the MDA-MB-231/FIC cell suspension subcutaneously into the back of 6-week-old male nude mice lacking T-cell function (BALB/cAJcl-nu/nu). The expressed CLuc is secreted into the blood from the cells and circulates in the living body. The blood containing CLuc can be drawn by using glass micro-hematocrit capillary tubes or a syringe 744013 1.13.12.6 additional information conjugation of prostaglandin E2 to luciferase by producing the N-hydroxysuccinimidyl ester of prostaglandin E2 by reaction of N-hydroxysuccinimde and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride and conjugation of the ester to luciferase (ratio 18/1) in 0.1 M potassium phosphate buffer, pH 7.2, with 0.15 M NaCl for 13 h on ice 699954 1.13.12.6 additional information construction of a cold-induced expression vector (pCold-ZZ-VL vector) in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of Staphylococcuss aureus protein A (ZZ-domain) and multiple cloning sites, fusing Cypridina luciferase to the ZZ-domain results in expression by Escherichia coli of a soluble cytoplasm enzyme but it is not bioluminescent (in contrast to other luciferases fused to the ZZ-domain) 696008 1.13.12.6 additional information establishment and validation of a yeast reporter assay, for detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system, using a secretory luciferase, Cypridina noctiluca luciferase CLuc, as an alternative to the conventional beta-galactosidase, the CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay, determination of the transcriptional activity of hundreds of yeast promoter fragments, overview 710855 1.13.12.6 additional information for large scale enzyme production, a simple production procedure is established using plant cell cultures. The plant cell culture tobacco BY-2 efficiently secretes luciferase, which is easily purified using a simple one-step ion-exchange chromatography method. The production yield is 20-30 mg of luciferase per liter of culture medium. The method offers a cost-effective production for Cypridina luciferase 746376 1.13.12.6 additional information wild-type and mutant enzymes show similar secretion efficiencies, and thermostabilities at 25-37°C, overview 745977 1.13.12.6 N182D site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar compared to the wild-type 745977 1.13.12.6 N182D/N404D site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type 745977 1.13.12.6 N182D/S406A site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type 745977 1.13.12.6 N404D site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant enzyme behaves similar to the wild-type 745977 1.13.12.6 S406A site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type 745977 1.13.12.6 T184A site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows increased protein accumulation compared to the wild-type 745977 1.13.12.6 T184A/N404D both N-glycosylation sites are mutated and lost 764716 1.13.12.6 T184A/N404D site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over 2.5fold increased protein accumulation compared to the wild-type 745977 1.13.12.6 T184A/S406A site-directed mutagenesis, mutation of a residue involved in a phoshorylation site, the mutant shows over slightly increased protein accumulation compared to the wild-type 745977