Quantitative Imaging of Retinoic Acid Activities in Living Mammalian Cells.

Retinoic acid (RA) is an intriguing metabolite that is necessary for embryonic development and differentiation in vertebrates. The present protocol demonstrates how to image RA activities indirectly in mammalian cells with ligand-activatable single-chain bioluminescence (BL) probes. We introduce 13 different molecular designs for characterizing an efficient single-chain probe that quantitatively visualizes RA activities with significant sensitivity. The key components included in the probes are (i) the N- and C-terminal fragments of artificial luciferase 16 (ALuc16), (ii) the ligand-binding domain of human retinoic acid receptor α (RAR LBD), and (iii) an LXXLL motif derived from common coactivators of nuclear receptors. The probe is highly selective and sensitive to all-trans-RA (at-RA) in animal cells. This protocol exemplifies quantitative imaging of the RA levels in serum and cerebrospinal fluid with a linear range in two orders. The present protocol is an important addition to conventional techniques on quantitative imaging of endogenous at-RA levels in live mammalian cells.

Molecular Tension Probe for In Vitro Bioassays.

Cell-free bioassays (CFBs) provide their own distinctive merits over cell-based bioassays (CBBs) including (i) rapid and on-site applicability, (ii) long-term utility, and (iii) bioanalytical versatility. The authors previously introduced a unique bioluminescent imaging probe for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. In this chapter, we exemplify that a full-length artificial luciferase is sandwiched between FRB (FKBP-rapamycin-binding domain of FKBP12-rapamycin-associated protein) and FKBP (FK506-binding protein) via minimal flexible linkers, named FRB-A23-FKBP. The rapamycin-activated PPIs between FRB and FKBP append molecular tension to the sandwiched luciferase, enhancing the enzymatic activity in a quantitative manner. The fusion protein, FRB-A23-FKBP, is three-step column-purified and the bioanalytical utility is characterized in various CFB conditions. This chapter guides the detailed protocols from the purification to the practical bioassays of FRB-A23-FKBP.

Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8+ T cells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector T cells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector T cells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

A New Lineage of Artificial Luciferases for Mammalian Cell Imaging.

The present protocol introduces a new lineage of artificial luciferases (ALucs) with unique optical properties for mammalian cell imaging. The primary candidate sequence was first created with a sequence logo generator, resulting in a total of 11 sibling sequences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in natural luciferase pools in public databases. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set. The protocol also exemplifies that the new lineage of ALucs was strongly luminescent in living mammalian cells with specific substrate selectivity to native coelenterazine. The success of this approach guides on how to engineer and functionalize marine luciferases for bioluminescence imaging and assays.

Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.

Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve.

BACKGROUND: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire. METHODS: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis. RESULTS: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model. CONCLUSION: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

Color-tunable bioluminescence imaging portfolio for cell imaging.

The present study describes a color-tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red-shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artificial Luciferase 16 (ALuc16) show 187 nm- and 105 nm-redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d. The optical properties of the new CTZ analogues were investigated such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to ALucs and are completely dark with RLuc derivatives, and 3d is highly specific to only NanoLuc. We further determined the theoretical background of the red-shifted BL maximum wavelengths (λBL) values according to the extended π conjugation of the CTZ backbone using Density Functional Theory (DFT) calculations. This color-tunable BL imaging system provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.

Therapy of Established Tumors with Rationally Designed Multiple Agents Targeting Diverse Immune-Tumor Interactions: Engage, Expand, Enable.

Immunotherapy of immunologically cold solid tumors may require multiple agents to engage immune effector cells, expand effector populations and activities, and enable immune responses in the tumor microenvironment (TME). To target these distinct phenomena, we strategically chose five clinical-stage immuno-oncology agents, namely, (i) a tumor antigen-targeting adenovirus-based vaccine (Ad-CEA) and an IL15 superagonist (N-803) to activate tumor-specific T cells, (ii) OX40 and GITR agonists to expand and enhance the activated effector populations, and (iii) an IDO inhibitor (IDOi) to enable effector-cell activity in the TME. Flow cytometry, T-cell receptor (TCR) sequencing, and RNA-sequencing (RNA-seq) analyses showed that in the CEA-transgenic murine colon carcinoma (MC38-CEA) tumor model, Ad-CEA + N-803 combination therapy resulted in immune-mediated antitumor effects and promoted the expression of costimulatory molecules on immune subsets, OX40 and GITR, and the inhibitory molecule IDO. Treatment with Ad-CEA + N-803 + OX40 + GITR + IDOi, termed the pentatherapy regimen, resulted in the greatest inhibition of tumor growth and protection from tumor rechallenge without toxicity. Monotherapy with any of the agents had little to no antitumor activity, whereas combining two, three, or four agents had minimal antitumor effects. Immune analyses demonstrated that the pentatherapy combination induced CD4+ and CD8+ T-cell activity in the periphery and tumor, and antitumor activity associated with decreased regulatory T-cell (Treg) immunosuppression in the TME. The pentatherapy combination also inhibited tumor growth and metastatic formation in 4T1 and LL2-CEA murine tumor models. This study provides the rationale for the combination of multimodal immunotherapy agents to engage, enhance, and enable adaptive antitumor immunity.

Highly sensitive eight-channel light sensing system for biomedical applications.

We demonstrate the potential of an eight-channel light sensing platform system, named Black Box I (BBI), for rapid and highly sensitive measurement of low-level light using a nonradioactive optical readout. We developed, normalized, and characterized the photon sensitivities of the eight channels of the BBI using placental alkaline phosphatase (PLAP) as a model imaging reporter. We found that the BBI system had a statistically strong linear correlation with the reference IVIS Lumina II system. When we applied normalization constants, we were able to optimize the photomultiplier tubes (PMT) of all eight channels of the BBI (up to r2 = 0.998). We investigated the biomedical utilities of BBI by: (i) determining alkaline phosphatase activities in mouse plasma samples as a diagnostic secretory biomarker of cancer, and (ii) diagnosing cancer metastases in the organs of mice bearing triple negative breast cancer. We provide an important new addition to low-cost biomedical instruments intended for pre-clinical diagnostic imaging with high sensitivity, high sample throughput, portability, and rapid on-site analysis of low-level light.

Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.

Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.

Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.

Molecular Imaging of Retinoic Acids in Live Cells Using Single-Chain Bioluminescence Probes.

Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was ∼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.

In vitro Determination of Rapamycin-triggered FKBP-FRB Interactions Using a Molecular Tension Probe.

As protein-protein interactions (PPI) have been mostly investigated in cellulo or in vivo, it is unclear whether the PPI-based imaging schemes are practically valid in a bioanalytical means in vitro. The present study exemplifies the PPI in vitro inside a unique single-chain probe, named TP2.4, which carries a full-length artificial luciferase (ALuc) sandwiched in between two model proteins of interest, e.g., FKBP and FRB, expressed in E. coli, and purified. We found that the TP2.4 efficiently recognizes its ligand in vitro and varies its molecular kinetics: i.e., rapamycin boosts the enzymatic affinities (Km) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (Kcat) and the maximal velocity (Vmax). The corresponding circular dichroism (CD) study shows that rapamycin weakly contributes to the enhancement of the α-helical contents in TP2.4. Kinetic constants according to the substrates revealed that a coelenterazine derivative, 6-N3-CTZ, exerted the best catalytic efficiency and the greatest variance in the total photon counts. The present study is the first in vitro example that demonstrates how intramolecular PPI works in a purified single-chain bioluminescent probe and what factors practically influence the biochemistry.

An IL-15 superagonist/IL-15Rα fusion complex protects and rescues NK cell-cytotoxic function from TGF-ß1-mediated immunosuppression.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a fundamental role in the immunosurveillance of cancers. NK cells of cancer patients exhibit impaired function mediated by immunosuppressive factors released from the tumor microenvironment (TME), such as transforming growth factor (TGF)-ß1. An interleukin (IL)-15 superagonist/IL-15 receptor α fusion complex (IL-15SA/IL-15RA; ALT-803) activates the IL-15 receptor on CD8 T cells and NK cells, and has shown significant anti-tumor activity in several in vivo studies. This in vitro study investigated the efficacy of IL-15SA/IL-15RA on TGF-ß1-induced suppression of NK cell-cytotoxic function. IL-15SA/IL-15RA inhibited TGF-ß1 from decreasing NK cell lysis of four of four tumor cell lines (H460, LNCap, MCF7, MDA-MB-231). IL-15SA/IL-15RA rescued healthy donor and cancer patient NK cell-cytotoxicity, which had previously been suppressed by culture with TGF-ß1. TGF-ß1 downregulated expression of NK cell-activating markers and cytotoxic granules, such as CD226, NKG2D, NKp30, granzyme B, and perforin. Smad2/3 signaling was responsible for this TGF-ß1-induced downregulation of NK cell-activating markers and cytotoxic granules. IL-15SA/IL-15RA blocked Smad2/3-induced transcription, resulting in the rescue of NK cell-cytotoxic function from TGF-ß1-induced suppression. These findings suggest that in addition to increasing NK cell function via promoting the IL-15 signaling pathway, IL-15SA/IL-15RA can function as an inhibitor of TGF-ß1 signaling, providing a potential remedy for NK cell dysfunction in the immunosuppressive tumor microenvironment.

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for chordoma.

Different epigenetic mechanisms of ERα implicated in the fate of fulvestrant-resistant breast cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα), which plays critical roles in breast cancer development. Fulvestrant has been effectively used to treat ERα-positive breast cancer, although resistance remains a critical problem. To elucidate the mechanism of resistance to fulvestrant, we established fulvestrant-resistant cell-lines named MFR (MCF-7 derived fulvestrant resistance) and TFR (T-47D derived fulvestrant resistance) from the ERα-positive luminal breast cancer cell lines MCF-7 and T-47D, respectively. Both fulvestrant-resistant cell lines lost sensitivity to estrogen and anti-estrogens. We observed diminished ERα expression at both the protein and mRNA levels. To address the mechanism of gene expression regulation, we examined epigenetic alteration, especially the DNA methylation level of ERα gene promoters. MFR cells displayed high methylation levels upstream of the ERα gene, whereas no change in DNA methylation was observed in TFR cells. Hence, we examined the gene expression plasticity of ERα, as there are differences in its reversibility following fulvestrant withdrawal. ERα gene expression was not restored in MFR cells, and alternative intracellular phosphorylation signals were activated. By contrast, TFR cells exhibited plasticity of ERα gene expression and ERα-dependent growth; moreover, these cells were resensitized to estrogen and anti-estrogens. The difference in epigenetic regulation among individual cells might explain the difference in the plasticity of ERα expression. We also identified an MFR cell-activating HER/Src-Akt/MAPK pathway; thus, the specific inhibitors effectively blocked MFR cell growth. This finding implies the presence of multiple fulvestrant resistance mechanisms and suggests that the optimal therapies differ among individual tumors as a result of differing epigenetic mechanisms regulating ERα gene expression.

An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

Nutritional status of patients with phenylketonuria in Japan.

Accumulating evidence suggests that hyperphenylalaninemia in phenylketonuria (PKU) can cause neuropsychological and psychosocial problems in diet-off adult patients, and that such symptoms improve after resumption of phenylalanine-restricted diet, indicating the need for lifetime low-phenylalanine diet. While limiting protein intake, dietary therapy should provide adequate daily intake of energy, carbohydrates, fat, vitamins, and microelements. We evaluated nutrient balance in 14 patients with classical PKU aged 4-38 years. Approximately 80-85% of the recommended dietary allowance (RDA) of protein in Japanese was supplied through phenylalanine-free (Phe-free) milk and Phe-free amino acid substitutes. Nutritional evaluation showed that the calorie and protein intakes were equivalent to the RDA. Phenylalanine intake was 9.8 ± 2.2 mg/kg of body weight/day, which maintained normal blood phenylalanine concentration by the 80% Phe-free protein rule. The protein, fat, and carbohydrate ratio was 9.5:23.9:66.6% with relative carbohydrate excess. Phe-free milk and amino acid substitutes provided 33.7% of carbohydrate, 82.1% of protein, and 66.7% of fat intake in all. Selenium and biotin intakes were 25.0% and 18.1% of the RDA and adequate intake (AI) for Japanese, respectively; both were not included in Phe-free milk. PKU patients showed low serum selenium, low urinary biotin, and high urinary 3-hydroxyisovaleric acid in this study. The intakes of magnesium, zinc, and iodine were low (71.5%, 79.5%, and 71.0% of the RDA, respectively) and that of phosphorus was 79.7% of the AI, although they were supplemented in Phe-free milk. PKU patients depend on Phe-free milk and substitutes for daily requirement of microelements and vitamins as well as protein and fat. Development of low-protein food makes it possible to achieve the aimed phenylalanine blood level, but this lowers the intake of microelements and vitamins from natural foods. The dietary habits vary continuously with age and environment in PKU patients. We recommend the addition of selenium and biotin to Phe-free milk in Japan and the need to review the composition of microelements and vitamins in A-1 and MP-11 preparations.

How to Fabricate Functional Artificial Luciferases for Bioassays.

The present protocol introduces fabrication of artificial luciferases (ALuc(®)) by extracting the consensus amino acids from the alignment of copepod luciferase sequences. The made ALucs have unique sequential identities that are phylogenetically distinctive from those of any existing copepod luciferase. Some ALucs exhibited heat stability, and strong and greatly prolonged optical intensities. The made ALucs are applicable to various bioassays as an optical readout, including live cell imaging, single-chain probes, and bioluminescent tags of antibodies. The present protocol guides on how to fabricate a unique artificial luciferase with designed optical properties and functionalities.

Fabrication of Molecular Strain Probes for Illuminating Protein-Protein Interactions.

A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for ligand-activated PPIs.