Light-emitting Proteins Research Articles

COELENTERAZINE-DEPENDENT LUCIFERASES: PROPERTIES AND APPLICATION

The review presents data on the structure of bioluminescent systems of marine animals that use coelenterazine-dependent luciferases as light-emitting proteins. It has been shown that due to the availability, stability and high quantum yield of reactions, these luciferases, as well as their genetic variants with new useful properties, are successfully applied as reporter molecules in a variety of analytical systems in vitro and in vivo. Their use provides high sensitivity, simple design and fast analysis.

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

Luciferase-Based Detection of Antibodies for the Diagnosis of HPV-Associated Head and Neck Squamous Cell Carcinoma.

Point-of-care tests are needed for the screening of head and neck squamous cell carcinoma (HNSCC) and other malignancies. Luciferase immunoprecipitation systems (LIPS), employing light-emitting proteins, were used to examine serum antibodies against several cancer-associated targets in blood donor controls and subjects with colon cancer (CC) and HNSCC. The assessment of antibodies against the wild type p53 tumor antigen showed that approximately 25% of the CC and 20% of the HNSCC patients were seropositive. In addition, humoral responses against two p53 mutants, p53-R175H and p53-R273H, generally tracked the antibody responses seen against wild type p53. Analysis of antibodies against highly specific biomarkers of HPV-16-associated malignancy, E2, E6, and E7 oncoproteins, revealed no seropositivity in blood donors and CC patients. However, 45% (9/20) of the HNSCC patients showed E6 seropositivity, which overlapped all the detectable E2 (40%; 8/20) and E7 seropositive subjects (35%; 7/20). Using neodymium magnets, ultrarapid LIPSTICKS testing of HPV-16 E6 antibodies in <60 s per HNSCC sample demonstrated almost the same diagnostic performance (40% sensitivity and 100% specificity) as LIPS testing in 2.5 h. While additional improvements and standardization are needed, these results highlight the possibility of using these approaches for the diagnosis of HPV-16-associated HNSCC.

Recombinant Hybrid Proteins as Biospecific Reporters for Bioluminescent Microassay

Light-emitting proteins, luciferases and photoproteins, are widely used as reporters in high sensitive binding assays in vitro and in vivo. The combination of luciferase and biospecific molecule with affinity to a certain target (immunoglobulin, oligonucleotide, hapten, etc.) promotes specificity of the assay. The molecules of the kind (biospecific bioluminescent labels) are usually obtained by chemical synthesis using different commercial reagents. However, this approach cannot provide homogeneity of the product; it causes the loss (sometimes essential) of specific activity of the initial molecules. As a rule chemical binding requires a thorough verification of the reaction conditions (balance of reagents, pH and temperature of the media, reaction duration, etc.) that would decrease a negative impact of the reagents and improve the yield of conjugates. Another way to obtain combination of the kind is the creation of a hybrid protein by using genetic engineering approach. In this case a genetic construction containing, in one frame, genes coding biospecific and reporter proteins as well as sequences, coding a linker between them and sometimes auxiliary polypeptides, is created. Gene fusion approach provides easy bacterial production, hybrids homogeneity, easy purification using auxiliary polypeptides etc. The paper describes several hybrids “luciferase–biospecific polypeptide” designed in the photobiology lab of the Institute of Biophysics SB RAS, their construction, production and application in microassay as biospecific bioluminescent probes

Better to light a candle than curse the darkness: illuminating spatial localization and temporal dynamics of rapid microbial growth in the rhizosphere.

The rhizosphere is a hotbed of microbial activity in ecosystems, fueled by carbon compounds from plant roots. Basic questions about the location and dynamics of plant-spurred microbial growth in the rhizosphere are difficult to answer with standard, destructive soil assays mixing a multitude of microbe-scale microenvironments in a single, often sieved, sample. Soil microbial biosensors designed with the luxCDABE reporter genes fused to a promoter of interest enable continuous imaging of the microbial perception of (and response to) environmental conditions in soil. We used the common soil bacterium Pseudomonas putida KT2440 as host to plasmid pZKH2 containing a fusion between the strong constitutive promoter nptII and luxCDABE (coding for light-emitting proteins) from Vibrio fischeri. Experiments in liquid media demonstrated that high light production by KT2440/pZKH2 was associated with rapid microbial growth supported by high carbon availability. We applied the biosensors in microcosms filled with non-sterile soil in which corn (Zea mays L.), black poplar (Populus nigra L.), or tomato (Solanum lycopersicum L.) was growing. We detected minimal light production from microbiosensors in the bulk soil, but biosensors reported continuously from around roots for as long as six days. For corn, peaks of luminescence were detected 1–4 and 20–35 mm along the root axis behind growing root tips, with the location of maximum light production moving farther back from the tip as root growth rate increased. For poplar, luminescence around mature roots increased and decreased on a coordinated diel rhythm, but was not bright near root tips. For tomato, luminescence was dynamic, but did not exhibit a diel rhythm, appearing in acropetal waves along roots. KT2440/pZKH2 revealed that root tips are not always the only, or even the dominant, hotspots for rhizosphere microbial growth, and carbon availability is highly variable in space and time around roots.

Synthesis of Novel BRET/FRET Protein Probes Containing Light-Emitting Proteins and Fluorescent Nonnatural Amino Acids

Abstract Novel light-emitting protein probes were designed and synthesized by incorporating fluorescent nonnatural amino acids and fusing light-emitting proteins. Gaussia luciferase was fused as a BRET donor to the C-terminus of maltose-binding protein, and BODIPY558–aminophenylalanine was incorporated into Tyr210, which is located near the maltose-binding site of maltose-binding protein in response to a four-base codon (CGGG). In addition to the luminescence of luciferase, the double-labeled protein showed the emission of BODIPY558, which was strongly quenched by a neighboring tryptophan in the absence of maltose but was recovered in the presence of maltose. The emission intensity ratio of luciferase and BODIPY558 increased 4.0-fold upon the addition of maltose. Similar ligand-dependent emission changes were observed for a translation reaction mixture expressing the double-labeled protein, indicating that the BRET protein probe was detectable in the presence of excess fluorescent molecules. BODIPY558-containing maltose-binding protein fused with GFP in place of luciferase was also synthesized and showed the FRET and maltose-dependent fluorescence of BODIPY558. Novel BRET/FRET protein probes synthesized by fusing light-emitting proteins and incorporating fluorescent nonnatural amino acids will become valuable tools for the bioimaging and diagnostic detection of specific ligands.

Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems

B-cell-mediated humoral responses are triggered in many human diseases, including autoimmune diseases, cancer, and neurologic and infectious diseases. However, the full exploitation of the information contained within a patient’s antibody repertoire for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed luciferase immunoprecipitation systems (LIPS) that harnesses light-emitting proteins to generate high-definition antibody profiles that are optimal for both diagnostics and biomarker discovery. Here, we describe the results and implications from a range of LIPS-antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis and offer a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring.

Orally Administered Bifidobacteria as Vehicles for Delivery of Agents to Systemic Tumors

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.

Real-time Monitoring of Vaccinia Virus Infection in Cultured Cells Using Light-emitting Proteins

Real-time Monitoring of Vaccinia Virus Infection in Cultured Cells Using Light-emitting Proteins

Two Dimensional Patterning of Fluorescent Proteins in Hydrogels

This work describes the successful micropatterning of hybrid systems consisting of hydrogel-dispersed optically active and controllable proteins on solid surfaces without degradation of the photophysical properties of the light-emitting biomolecules. It demonstrates the preservation of the luminescence properties of proteins entrapped into isolated microstructures of poly(acrylamide) gel. This way we can exploit both the structural and function-preserving properties of the hydrogels and the functionality of light-emitting proteins. We believe that this approach can open the way to the realization of nanopatterned optical memories based on photochromic biomolecules.

Correction: Corrigendum: Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins

Nat. Biotechnol. 22, 313–320 (2003) On page 319, column 1, line 9 from bottom, the phrase “subcutaneous colon fibrosarcoma” should have read “subcutaneous fibrosarcoma.”

Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals.

Early detection of tumors and their metastases is crucial for the prognosis of cancer treatment. Traditionally, tumor detection is achieved by various methods, including magnetic resonance imaging and computerized tomography. With the recent cloning, cellular expression, and real-time imaging of light-emitting proteins, such as Renilla luciferase (Ruc), bacterial luciferase (Lux), firefly luciferase (Luc), green fluorescent protein (GFP), or Ruc-GFP fusion protein, significant efforts have been focused on using these marker proteins for tumor detection. It has also been demonstrated that certain bacteria, viruses, and mammalian cells (BVMC), when administered systemically, are able to gain entry and replicate selectively in tumors. In addition, many tissue/tumor specific promoters have been cloned which allow transgene expression specifically in tumor tissues. Therefore, when light-emitting protein encoded BVMC are injected systemically into rodents, tumor-specific marker gene expression is achieved and is detected in real time based on light emission. Consequently, the locations of primary tumors and previously unknown metastases in animals are revealed in vivo. In the future it will likely be feasible to use engineered light-emitting BVMC as probes for tumor detection and as gene-delivery vehicles in vivo for cancer therapy.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Elimination of internal restriction enzyme sites from a bacterial luminescence (luxCDABE) operon.

BioTechniquesVol. 24, No. 1 BenchmarksOpen AccessElimination of Internal Restriction Enzyme Sites from a Bacterial Luminescence (luxCDABE) OperonChristine R. Voisey & Ferenc MarincsChristine R. VoiseyAgResearch, Grasslands Research Center, Palmerston North, New ZealandSearch for more papers by this author & Ferenc Marincs*Address correspondence to Dr. Ferenc Marincs, AgResearch, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand. Internet: E-mail Address: marincsf@agresearch.cri.nzAgResearch, Grasslands Research Center, Palmerston North, New ZealandSearch for more papers by this authorPublished Online:15 Aug 2018https://doi.org/10.2144/98241bm11AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Elimination of Internal Restriction Enzyme Sites from a Bacterial Luminescence (luxCDABE) Operon." , 24(1), pp. 56–58FiguresReferencesRelatedDetailsCited ByBiotinylated Bioluminescent Probe for Long Lasting Targeted in Vivo Imaging of Xenografted Brain Tumors in Mice5 June 2017 | ACS Chemical Neuroscience, Vol. 9, No. 1Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain9 December 2014 | Microbial Biotechnology, Vol. 8, No. 1Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria1 January 2008 | Biotechnology and Bioengineering, Vol. 100, No. 3A High-Throughput, Homogeneous, Bioluminescent Assay for Pseudomonas aeruginosa Gyrase Inhibitors and Other DNA-Damaging AgentsSLAS Discovery, Vol. 12, No. 6An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express α-fetoprotein6 January 2006 | Cancer Gene Therapy, Vol. 13, No. 8Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins8 February 2004 | Nature Biotechnology, Vol. 22, No. 3Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals19 July 2003 | Analytical and Bioanalytical Chemistry, Vol. 377, No. 6Integration-Proficient Pseudomonas aeruginosa Vectors for Isolation of Single-Copy Chromosomal lacZ and lux Gene FusionsAnna Becher & Herbert P. Schweizer29 August 2018 | BioTechniques, Vol. 29, No. 5 Vol. 24, No. 1 Follow us on social media for the latest updates Metrics Downloaded 140 times History Published online 15 August 2018 Published in print January 1998 Information© 2018 Author(s)PDF download