Luciferase NLuc Site-Specific Conjugation to Generate Reporters for In Vitro Assays.

NanoLuc (NLuc) is an artificial coelenterazine-dependent luciferase generated from the deep-sea shrimp Oplophorus gracilirostris. Its peculiar properties─small size and long-lasting bright bioluminescence triggered with the synthetic substrate furimazine─have made this enzyme popular as a reporter in a variety of analytical systems. Basically, to ensure the assay specificity, NLuc is genetically fused to the polypeptide with affinity for the corresponding target. The approach, however, has a limitation for non-protein biospecific molecules, thus forcing the production of biospecific luciferase derivatives via chemical conjugation. Unfortunately, it yields a heterogeneous product and often results in the loss of a significant part of bioluminescence activity. Here, we report on NLuc site-directed conjugation by combining these two approaches: several luciferase derivatives, genetically extended with hexapeptides carrying a unique Cys residue, were obtained, and the variant with activity equal to that of the intact NLuc was found. Biospecific molecules of the most commonly used types (low-weight hapten, oligonucleotide, antibody, and DNA aptamer) were chemically attached to this NLuc variant through the unique Cys using an orthogonal conjugation approach. The resulting conjugates were tested as labels in the bioluminescence assay and were shown to detect the corresponding molecular targets (e.g., cardiac markers) with high sensitivity.

Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features.

Ca2+-triggered coelenterazine-binding protein (CBP) is a natural form of the luciferase substrate involved in the Renilla bioluminescence reaction. It is a stable complex of coelenterazine and apoprotein that, unlike coelenterazine, is soluble and stable in an aquatic environment and yields a significantly higher bioluminescent signal. This makes CBP a convenient substrate for luciferase-based in vitro assay. In search of a similar substrate form for the luciferase NanoLuc, a furimazine-apoCBP complex was prepared and verified against furimazine, coelenterazine, and CBP. Furimazine-apoCBP is relatively stable in solution and in a frozen or lyophilized state, but as distinct from CBP, its bioluminescence reaction with NanoLuc is independent of Ca2+. NanoLuc turned out to utilize all the four substrates under consideration. The pairs of CBP-NanoLuc and coelenterazine-NanoLuc generate bioluminescence with close efficiency. As for furimazine-apoCBP-NanoLuc pair, the efficiency with which it generates bioluminescence is almost twice lower than that of the furimazine-NanoLuc. The integral signal of the CBP-NanoLuc pair is only 22% lower than that of furimazine-NanoLuc. Thus, along with furimazine as the most effective NanoLuc substrate, CBP can also be recommended as a substrate for in vitro analytical application in view of its water solubility, stability, and Ca2+-triggering "character".

N-extended photoprotein obelin to competitively detect small protein tumor markers.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

Starch-Coated Magnetic Iron Oxide Nanoparticles for Affinity Purification of Recombinant Proteins.

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100-500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications.

: The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate-coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization-Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme-substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.

RNA aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis.

Nowadays, there are no specific laboratory tests for establishing the diagnosis of multiple sclerosis (MS). The presence of proteolytic autoantibodies against myelin basic protein is now considered as a characteristic feature of MS. New 2'-F-containing RNA aptamer of high affinity and specificity to these antibodies was selected. Covalent conjugate of this aptamer and Ca(2+)-regulated photoprotein obelin was obtained for the first time and applied as a label in bioluminescent microplate assay to detect target antibodies. The developed model solid-phase microassay is simple, fast, and highly sensitive.

Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules.

Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix. Whereas, deletion or substitution of the C-terminal proline could noticeably change the bioluminescence activity, stability or the yield of an active photoprotein complex. Therefore, modifications of the first α-helix Arg has a clear destructive effect on the main photoprotein properties. A C-terminal hydrogen-bond network is proposed to be important for the stability of photoprotein molecules towards external disturbances, when taking part in the formation of locked protein conformations and isolation of coelenterazine-binding cavities.

Bioluminescent aptamer-based microassay for detection of melanoma inhibitory activity protein (MIA).

Melanoma inhibitory activity protein (MIA) does obviously offer the potential to reveal clinical manifestations of melanoma. Despite a pressing need for effective diagnosis of this highly fatal disease, there are no clinically approved MIA detection ELISA kits available. A recommended MIA threshold has not yet been defined, mostly by reason of variability in immunoglobulins' affinity and stability, the difference in sample preparation and assay conditions. Here we present a pair of high-affinity DNA aptamers developed as an alternative recognition and binding element for MIA detection. Their stability and reproducible synthesis are expected to ensure this analysis under standard conditions. The devised aptamer-based solid-phase microassay of model standard and control human sera involves luciferase NLuc as a highly sensitive reporter. Bioluminescence dependence on MIA concentration ranges in a linear manner from 2.5 to 250 ng mL-1, providing a MIA detection limit of 1.67 ± 0.57 ng mL-1.

Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.

Ca(2+)-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor-acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His-Trp-Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His-Trp-Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.

Simultaneous bioluminescent immunoassay of serum total and IgG-bound prolactins.

Novel dual-analyte single-well bioluminescence immunoassay (BLIA) for total and IgG-bound prolactins was developed on the base of Ca(2+)-regulated photoprotein obelin mutants with altered color and kinetics of bioluminescence signal as reporters. The mutants W92F-H22E and Y138F were chemically conjugated with monoclonal mouse anti-hPRL and anti-hIgG immunoglobulins and thus displayed signals from total prolactin and IgG-bounded prolactin (macroprolactin) correspondingly. Bioluminescence of the reporters was simultaneously triggered by a single injection of Ca(2+) solution and discriminated via bioluminescent signal spectral and time resolution. The developed microplate-based immunoassay allows detection of two prolactin forms in crude serum without additional manipulations (e.g., gel chromatography or PEG-precipitation). Total prolactin bioluminescence immunoassay in standard, control, and clinical sera offers high sensitivity and reproducibility. The BLIA results show good correlation with those obtained by RIA and immunoassay after gel chromatography.

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein.

The bioluminescent systems of many marine organisms are comprised of two proteins--the Ca(2+)-regulated photoprotein and green-fluorescent protein (GFP). This work reports the cloning of the full-size cDNA encoding GFP (cgreGFP) from jellyfish Clytia gregaria, its expression and properties of the recombinant protein. The overall degree of identity between the amino acid sequence of the novel cgreGFP and the sequence of GFP (avGFP) from Aequorea victoria is 42% (similarity--64%) despite these GFPs originating from jellyfish that both belong to the same class, Hydrozoa. However although the degree of identity is low, three residues, Ser-Tyr-Gly, which form the chromophore are identical in both GFPs. The cgreGFP displayed two absorption peaks at 278 and 485 nm, and the fluorescence maximum at 500 nm. The fluorescence quantum yield was determined to be 0.86, the brightness to be 54 mM(-1) cm(-1). For the first time we have also demonstrated an efficient radiationless energy transfer in vitro between clytin and cgreGFP in solution at micromolar concentrations. The cgreGFP may be a useful intracellular fluorescent marker, as it was able to be expressed in mammalian cells.

Ca2+-regulated photoproteins: effective immunoassay reporters.

Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are "self-contained", triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element--bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects.

The Ca2+ -Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation.

Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the specific aptamers. The assay involves Ca2+ -regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.

Bioluminescent aptamer-based solid-phase microassay to detect lung tumor cells in plasma.

Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5' end of both aptamers carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p < 0.001). The area under ROC curve with the value of 0.901 distinguishes well between the two groups under investigation.

Bioluminescent aptamer-based sandwich-type assay of anti-myelin basic protein autoantibodies associated with multiple sclerosis.

Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2'-F-Py RNA aptamers against the target autoantibodies as biospecific elements, and Ca2+-regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests.

Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay.

Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max) = 390 nm) and the mutant Y139F emits greenish light (lambda(max) = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca(2+) solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.

Bioluminescent SNP genotyping technique: Development and application for detection of melanocortin 1 receptor gene polymorphisms.

SNP genotyping based on the reaction of specific primer extension with the following bioluminescent detection of its products was shown to be potentially applicable for biomedical exploration. The paper describes its elaboration and first application in extensive biomedical research concerning MC1R gene variants' frequency and associations with clinical characteristics in melanoma patients of Eastern Siberia (Krasnoyarsk region, Russia). Polymorphisms rs 1805007 (R151C), rs 1805008 (R160W), and rs 1805009 (D294H) were detected in 174 DNA samples from patients with histologically proved diagnosis of cutaneous melanoma and in 200 samples from healthy individuals. All the results on bioluminescent SNP genotyping were confirmed by Sanger sequencing. Some features characteristic of the population were found, i.e. melanoma is mostly associated with R160W or R151C while variant D294H is extremely rare; simultaneous carriage of any two investigated variants is also strongly associated with melanoma; R151C is associated with ulceration and consequently the disease course is more aggressive, etc. The design of the technique allows fast evaluation of any known diagnostically important SNP frequencies and associations across population.

Hybrid Minimal Core Streptavidin-Obelin as a Versatile Reporter for Bioluminescence-based Bioassay.

Ca2+ -regulated photoprotein obelin was genetically fused with a minimum-sized core streptavidin. Hybrid protein (SAV-OL) was produced by bacterial expression and applied as a specific bioluminescent probe in diverse solid-phase assays. The obtained results clearly demonstrate specific activity of each domain indicating its proper folding with favorable space orientation. SAV-OL has been shown to be a much more sensitive label than the chemical conjugate of a full-length streptavidin with obelin.

Mutants of Ca2+ -regulated Photoprotein Obelin for Site-specific Conjugation.

Color variants of Ca2+ -regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample. Availability of SH-groups, accessible for chemical modification, essentially simplifies the synthesis of biospecific conjugates, increases their yield and conserves obelins' bioluminescence activity. Conjugates with immunoglobulin and oligonucleotide were produced and successfully applied in single nucleotide polymorphism genotyping.