A dihydrofolate reductase-deficient Chinese hamster ovary (CHO-K1/dhfr-) cell line stably expressing Gaussia luciferase with a histidine-tag sequence at the carboxyl terminus (GLase-His) was established. Recombinant GLase-His was purified from serum-containing culture medium by single-step Ni-chelate column chromatography in the presence of 2 M NaCl and 0.01% Tween 20. The protein yield of GLase-His with over 95% purity was 0.5 mg from 0.9 L of the cultured medium. The enzymatic properties of purified GLase-His were characterized. Interestingly, non-ionic detergent Tween 20 stabilized and stimulated GLase-His activity and its luminescence activity was stimulated 2-fold by the synergistic effect of 0.01% Tween 20 and 150 mM NaCl.
DOI : 10.1016/j.pep.2017.09.001 Anahtar Kelimeler :
CHO-K1/dhfr-, Ni-chelate column, Non-ionic detergent, Coelenterazine, GLase, Gaussia luciferase, GLase-His, histidine-tagged Gaussia luciferase, CHO, Chinese hamster ovary, dhfr, dihydrofolate reductase, Imax, maximum intensity of luminescence, rlu, relative light units, PBS, phosphate-buffered saline (10 mM phosphate buffer (pH 7.6), 2.7 mM KCl, and 137 mM NaCl), PBSTE, PBS containing 0.01% Tween 20 and 10 mM EDTA, E/S, enzyme-to-substrate ratio
Cilt: 141 Sayı: 0 Sayfa: 32-38 ISSN: 1046-5928
A method for identifying modified lysine residues in a protein, using lysine-specific endopeptidase treatment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping, is described. As a model protein, the photoprotein aequorin was chosen and the N-hydroxysuccinimide ester of biotin was employed to chemically modify the lysine residues. After digestion with lysine-specific endopeptidase, the biotinylated residues of an amino terminus and five potential lysine residues were identified by MALDI-TOF-MS without any other separation procedure.
A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20 mg from 2 L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca2+ sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca2+. Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.
Recombinant apoaequorin expressed in the periplasmic space ofEscherichia colicells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25–35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed inE. colicells.
The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35 kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6 M urea and purified by urea–nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6 M urea could be refolded rapidly by dilution with 50 mM Tris–HCl (pH 7.8)–10 mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1 μM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.
We constructed a vector for soluble protein expression in the cytoplasm of Escherichia coli cells using the cold induced system. The vector, named pCold-ZZ-P-X, consists of a histidine tag sequence, IgG binding domain of protein A (ZZ domain), the cleavage site of human rhinovirus 3C protease followed by the multiple cloning sites under the controlled of the cold shock protein A (cspA) promoter and the lac operator. Using this expression vector, the calcium binding photoprotein mitrocomin from luminous jellyfish was successfully expressed as a soluble ZZ fusion protein and purified. After removing the ZZ domain by protease digestion, recombinant apomitrocomin was obtained and then regenerated to mitrocomin by incubation with coelenterazine. The luminescence properties of recombinant mitrocomin were characterized and compared to other photoproteins including aequorin, clytin-I and clytin-II.
The mutated recombinant aequorin with a reactive cysteine residue (Cys–aequorin) was highly purified and then conjugated with a maleimide-activated antibody without significant loss of luminescence activity. The conjugate ratio of Cys–aequorin to heavy chain of immunoglobulin G (IgG) was estimated to be 1:1. To test the bioluminescent immunoassay with aequorin-labeled antibody, α-fetoprotein (AFP), a serological marker of liver cancer, was used as a model analyte. The measurable range of AFP was 0.02 to 200 ng/ml with the coefficient of variation between 2.1 and 4.5%.
Highly purified histidine-tagged aequorin with a reactive cysteine residue (His-Cys4-aequorin) was obtained from the periplasmic space of Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The procedure yielded 40.3 mg of His-Cys4-aequorin from 2 L of cultured cells with over 95% purity. The chemical conjugates of His-Cys4-aequorin with maleimide-acitivated streptavidin and maleimide-activated biotin were prepared without significant loss of luminescence activity and were applied to the bioluminescent sandwich immunoassay for α-fetoprotein (AFP) as a model analyte. The measurable range of AFP by these conjugates was 0.01–100 ng/ml and the sensitivities were similar to that using aequorin-labeled specific antibody and amino-biotinylated aequorin.
DOI : 10.1016/j.pep.2012.04.001 Anahtar Kelimeler :
Chemical conjugation, Maleimide biotin, α-Fetoprotein
Cilt: 83 Sayı: 2 Sayfa: 205-210 ISSN: 1046-5928
Two luciferase genes (dPaLuc and vPaLuc) and one paralogue of luciferase (PaLL) were isolated from the Panamanian luminous click beetle, Pyrophorus angustus (Elateridae, Pyrophorinae). The transcripts of dPaLuc and vPaLuc were predominantly detected in the body parts with dorsal photophore and ventral photophore, respectively, and the transcript of PaLL was detected in both parts. The gene products of dPaLuc and vPaLuc possessed luminescence activity with firefly luciferin (λmax = 536 and 566 nm, respectively) but did not show significant activity of fatty acyl-CoA synthesis. On the other hand, the gene product of PaLL had fatty acyl-CoA synthetic activity with very weak luminescence activity. The catalytic properties of click beetle luciferase are different from our previous results that firefly luciferase has both luminescence activity and fatty acyl-CoA synthetic activity. These results suggested that the ancestral fatty acyl-CoA synthetase in the Pyrophorinae lineage has undergone gene duplication event, followed by specialization of one copy in luciferase. Subsequently, the luciferase was duplicated again and the two copies diverged in their luminescent color and expression pattern.
To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis. All of the sulfate cellufine-retarded peptide fragments gave the same N-terminal amino acid sequence, which started at residue-68 of human follistatin, suggested that those fragments starting from residue-68 contain the heparin binding site. The multiple fragments might represent the oxidized, non-glycosylated or glycosylated forms of follistatin(68–113) resulting from the V8 digestion.
We have developed a method of video rate bioluminescence imaging to investigate protein secretion from a single mammalian cell and analyzed the localization, secretory frequency, and quantification of secreted protein. By detecting the luminescence signals from the Gaussia luciferase (GLase) reaction using a high-speed electron-multiplying charge-coupled device (EM-CCD) camera, video rate imaging was performed with a time resolution within 500 ms/image over 30 min in living cells. As a model study, we applied the method to visualize the glucose-stimulated insulin secretion from clustered pancreatic MIN6 β cells using the fused protein of GLase with preproinsulin. High-quality video images clearly showed that the glucose-stimulated insulin secretion from the clustered MIN6 β cells oscillated within a period of a few minutes over 10 min. In addition, the glibenclamide-induced insulin secretion from the clustered MIN6 β cells was visualized, suggesting that bioluminescence video rate imaging is a useful method for validating drug action in living cells.
A homologous gene of beetle luciferase, AbLL (Agrypnus binodulus luciferase-like gene) was isolated from a Japanese non-luminous click beetle, A. binodulus, and its gene product was characterized. The identity of amino acid sequence deduced from AbLL with the click beetle luciferase from the Jamaican luminous click beetle, Pyrophorus plagiophthalmus, is 55%, which is higher than that between click beetle luciferase and firefly luciferase (∼ 48%). Phylogenetic analysis indicated that AbLL places in a clade of beetle luciferases, suggesting that AbLL is an orthologous gene of beetle luciferase. The gene product of AbLL (AbLL) has medium- and long-chain fatty acyl-CoA synthetase activity, but not luciferase activity. The fatty acyl-CoA synthetic activity was slightly inhibited in the presence of beetle luciferin, suggesting that AbLL has poor affinity for beetle luciferin. By comparing the amino acid residues of the catalytic domains in beetle luciferases with AbLL, the key substitutions for the luminescence activity in beetle luciferase will be proposed.
Two homologous genes of firefly luciferase, LcLL1 and LcLL2, were cloned from the Japanese firefly Luciola cruciata, and were expressed and characterized. The gene product of LcLL1 had long-chain fatty acyl-CoA synthetic activity, but not luciferase activity. The other gene product of LcLL2 did not show enzymatic activities of acyl-CoA synthetase and luciferase. RT-PCR analysis showed that the transcript of LcLL1 was abundant in larva but very low in adult, while LcLL2 was expressed in both larva and adult. Phylogenetic analysis indicated that LcLL1 and LcLL2 are paralogous genes of firefly luciferase. Recently, we found that CG6178 in Drosophila melanogaster is an orthologue of firefly luciferase and shows fatty acyl-CoA synthetic activity, but not luciferase activity. These results suggest that firefly luciferase might be evolved from a fatty acyl-CoA synthetase by gene duplication in insects.
A high-molecular-size plasmid (37-kb), named pNO33, has been isolated from Streptomyces albulus IFO14147, a producer of ε-poly-l-lysine which exhibited antimicrobial activity. The sequence analysis of a 4.6-kb fragment in pNO33 revealed four putative open reading frames, one of which exhibited a significant homology to the bldB gene product involved in morphogenesis and antibiotic production by S. coelicolor.
Feeding experiment to the marine ostracod crustacean, Cypridina (Vargula) hilgendorfii, using l-tryptophan labeled with deuterium at indole ring revealed that the labeled l-tryptophan was incorporated into Cypridina luciferin as a component and the animals were able to synthesize the luciferin in a de novo synthetic pathway.
In a luminous ostracod Cypridina (Vargula) hilgendorfii, Cypridina luciferin with an imidazopyrazinone structure (3,7-dihydroimidazopyrazin-3-one) is utilized for the luminescence reaction. To identify the biosynthetic units of Cypridina luciferin, the stable isotope labeled compounds were examined by feeding experiments with living Cypridina specimens. The incorporation of the labeled compounds into Cypridina luciferin was identified by the method of LC/ESI-TOF-MS analyses and these results suggested that l-tryptophan, l-arginine and l-isoleucine are structural units of Cypridina luciferin.
Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81–89% identities to each other and 60–81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545–546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550–568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.
Aequorin is a Ca2+-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O2 to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca2+ of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca2+ and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.
Aequorin is a luminescent protein present in the jellyfish Aequoria victoria which emits light (at 460 nm) in the presence of Ca2+. We report here that aequorin can be used as a reporter enzyme to monitor gene expression in eukaryotic cells. A cDNA encoding apoaequorin was fused to several eukaryotic promoters, including those of SV40, RSV and the HSV-1 tk gene, and introduced into several cell lines such as CV-1, COS and HeLa. At appropriate times after transfection, the aequorin activities in cell extracts were measured by monitoring the intensity of lighte mitted at 460 nm when triggered by Ca2+ by the use of a photomultiplier photometer. The aequorin assay was shown to be as sensitive as the conventional CAT assay, and the relative activities of various promoters estimated by the aequorin assay were in general agreement with those obtained by the CAT assay. The aequorin assay can be done within 6–7 h from the preparation of extract to the measurement of activity without using radioctive compounds.
DOI : 10.1016/0378-1119(90)90260-X Anahtar Kelimeler :
Bioluminescence, calcium ion, CAT assay, eukaryotic vectors, mammalian gene promoters, recombinant DNA, transfection, aaeq, gene (DNA) encoding apoaequorin, aeq, gene (DNA) encoding aequorin, bp, base pair(s), Cm, chloramphenicol, CAT, Cm acetyltransferase, HSV-1, Herpes simplex virus type I, IU, international unit(s), LTR, long terminal repeat, MT-I, gene encoding methallothionein I, nt, nucleotide(s), PBS(−), Ca2+ - and Mg2+ -free phosphate-buffered saline (8 g NaCl/0.2 g KCl/1.15 g Na2HPO4/0.02 g KH2PO4 per liter of distilled water), Pn, penicillin G potassium salt, rlu, relative light unit(s), RSV, Rous sarcoma virus, SV40, simian virus 40, tk, gene encoding thymidine kinase
Cilt: 96 Sayı: 2 Sayfa: 249-255 ISSN: 0378-1119
The bacterial cloning vector, pGreenscript A, derived from the mutated Aequorea green fluorescent protein (GFP-S65A) gene, when expressed in E. coli produced colonies that showed yellow color under daylight and strong green fluorescence under long-wave ultraviolet light. The vector was used to select for inserted foreign genes based on the loss of the yellow color/green fluorescence of E. coli cells caused by the insertional inactivation of GFP production.
Cypridina luciferin from the luminous ostracod Cypridina (Vargula) hilgendorfii has an imidazopyrazinone core structure (3,7-dihydroimidazopyrazin-3-one), which is identical to that of coelenterazine. Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) was isolated for the first time and the chemical structure was identified by LC/ESI–TOF–MS analysis. Furthermore, Cypridina luciferyl sulfate was chemically synthesized, and its absorption and MS/MS spectra were in agreement with that of Cypridina luciferyl sulfate isolated. Using the crude extracts of Cypridina specimens, Cypridina luciferyl sulfate could be converted to Cypridina luciferin in the presence of adenosine 3′,5′-diphosphate (PAP), and Cypridina luciferin was converted to Cypridina luciferyl sulfate in the presence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). These results suggested that a sulfotransferase catalyzes the reversible sulfation of Cypridina luciferin in Cypridina hilgendorfii. In aqueous solution, Cypridina luciferyl sulfate was more stable than Cypridina luciferin and might be a storage form of Cypridina luciferin.
We established a simultaneous bioluminescent assay utilizing aequorin (Aq) and biotinylated firefly luciferase (b-Luc); furthermore, we developed a highly sensitive and rapid tandem bioluminescent immunoassay (BLIA) involving the Aq-labeled Fab fragment and b-Luc–streptavidin complex. Minimum detection limits of Aq and b-Luc were 9.4 × 10−21 mol assay−1 (blank + 3S.D.) and 3.6 × 10−19 mol assay−1 (blank + 3S.D.), respectively. Measurements of two luminescent proteins were completed in 4 s with a single assay medium. In this study, prostatic acid phosphatase (PAP) and prostate specific antigen (PSA), which served as analytes, were measured in the tandem BLIA. PAP and PSA were detected by the Aq-labeled anti-Dig Fab fragment and b-Luc–streptavidin complex, respectively. The measurable ranges of PAP and PSA were 0.04–100 and 0.2–200 ng mL−1, respectively. This technique was also applied to the simultaneous measurement of PSA and α-fetoprotein (AFP). Measurable ranges of PSA and AFP were 0.2–200 and 1.95–1000 ng mL−1, respectively. Levels of PAP and PSA or PSA and AFP in human serum could be accurately determined with the proposed BLIA. Satisfactory correlations were observed between results obtained from the proposed BLIA and those derived from commercial kits.
Firefly luciferase is a member of the acyl-adenylate/thioester-forming superfamily of enzymes and catalyzes the oxidation of firefly luciferin with molecular oxygen to emit light. Knowledge of the luminescence mechanism catalyzed by firefly luciferase has been gathered, leading to the discovery of a novel catalytic function of luciferase. Recently, we demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis from fatty acids in the presence of ATP, Mg2+ and coenzyme A. Based on identification of fatty acyl-CoA genes in firefly, Drosophila, and non-luminous click beetles, we then proposed that the evolutionary origin of firefly luciferase is a fatty acyl-CoA synthetase in insects. Further, we succeeded in converting the fatty acyl-CoA synthetase of non-luminous insects into functional luciferase showing luminescence activity by site-directed mutagenesis.
A model compound, 4-(4-hydroxyphenyl)methylideneimidazol-5-one, undergoes a reversible redox reaction identical to that of the Aequorea green fluorescent protein (GFP), strongly suggesting that the GFP chromophore is derived via the autoxidation of a nonfluorescent dihydro precursor in dihydro-GFP.
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.
The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%) > RLase (8.0%) > RLase-547 (0.73%) > 19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases.
Simian virus 40 (SV40) virus-like particles (VLPs) are efficient nanocarriers for gene delivery. VLPs conjugated to human epidermal growth factor (hEGF) were prepared and the cell selectivity of the VLP was examined using human epithelial carcinoma A431 cells, which overexpress the EGF receptor. The endocytic efficiency was determined by the level of Gaussia luciferase activity from the encapsulated protein in hEGF-conjugated VLPs. EGF receptor-mediated endocytosis of hEGF-conjugated VLPs was significantly increased and was confirmed by fluorescence imaging using mCherry encapsulated in hEGF-conjugated VLPs. These results suggest that VLPs of SV40 conjugated to a specific ligand could be used for cell selective gene delivery.
DOI : 10.1016/j.jbiotec.2011.06.030 Anahtar Kelimeler :
SV40, simian virus 40, VLP, virus-like particle, hEGF, human epidermal growth factor, GLase, Gaussia luciferase, VLPN138C, N138C VP1 VLP, GV, GLase encapsulated in VLPN138C, VE, hEGF conjugated VLPN138C, GVE, GLase encapsulated in VLPN138C conjugating hEGF, mCherry-VLPN138C, mCherry encapsulated in VLPN138C, mCherry-VLPN138C-hEGF, mCherry encapsulated in VLPN138C conjugating hEGF, Cell selectivity, Drug delivery, Gaussia luciferase, mCherry
Cilt: 155 Sayı: 2 Sayfa: 251-256 ISSN: 0168-1656