Application of chromosomal gene insertion into Escherichia coli for expression of recombinant proteins.

Escherichia coli is the most popular organism used for producing recombinant proteins. However, the expression of recombinant proteins in E. coli sometimes results in the aggregation of proteins as an inclusion body in host cells. In such cases, it is necessary to optimize the refolding conditions to obtain the recombinant protein in its native form. Several techniques, such as reducing the concentration of the induction reagent during E. coli cultivation, have been developed to prevent the formation of inclusion bodies by controlling protein expression levels. In this study, we inserted one copy of a target gene under the control of T7 promoter into the E. coli chromosome using the Red-mediated recombination system. This system enabled soluble expression of the putative d-aminoacylase from Pyrococcus abyssi, which is expressed in an insoluble form following the use of conventional plasmid-based T7 promoter/polymerase systems. The relationship between the number of inserted gene copies and amount of soluble recombinant protein produced was evaluated by multiple insertions of the eGFP gene into the E. coli chromosome. The results revealed that the total expression from the insertion of one copy was around 1/5 that of the pET plasmid system and that expression increased as the inserted gene copy number increased up to five copies.

From metal-organic framework to intrinsically fluorescent carbon nanodots.

Highly photoluminescent carbon nanodots (CNDs) were synthesized for the first time from metal-organic framework (MOF, ZIF-8) nanoparticles. Coupled with fluorescence and non-toxic characteristics, these carbon nanodots could potentially be used in biosafe color patterning.

Enhanced beetle luciferase for high-resolution bioluminescence imaging.

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin alpha by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.

Current advanced bioluminescence technology in drug discovery.

BACKGROUND: Bioluminescence technology is based on the luciferin-luciferase reaction and is generally well known as a reporter gene assay system that uses firefly luciferase. It has revolutionized the field of transcriptional analysis owing to its usability and quantitative capability. Several methods for transcription analysis have emerged in the past two decades. Recently, novel bioluminescence techniques that differ from typical approaches were developed for the detection of transcriptional regulation or direct protein-protein interactions. OBJECTIVE: As each method has its own characteristics, this review summarizes the latest bioluminescence methods that are applicable to the field of drug discovery research. METHODS: Considering the diversity of related techniques, this review covers several aspects that have been divided into the following classes: variation of reporter gene assays, secretion properties, protein-protein interaction assays in living cells and bioluminescence imaging of living cells. RESULTS/CONCLUSIONS: The practical application of several luciferins and/or luciferases and the generation of novel applications by incorporating fluorescent molecules into bioluminescence techniques will become increasingly important because bioluminescence technology has a significant potential depending on how we use it.

14-3-3 regulates the nuclear import of class IIa histone deacetylases.

Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

The mobility of Bach2 nuclear foci is regulated by SUMO-1 modification.

The small ubiquitin-like modifier-1 (SUMO-1) modulates the functions of nuclear proteins by changing their structure and/or subnuclear localization. Several nuclear proteins form dynamic higher order nuclear structures, termed non-chromatin nuclear domains, which are involved in the regulation of nuclear function. However, the role that SUMO modification of the component proteins plays in the regulation of the activity and function of nuclear domains is unclear. Here we demonstrate that nuclear domains formed by Bach2, a transcription repressor, show restricted movement and undergo fusion events upon oxidative stress. Mutation of the SUMO-acceptor lysines in Bach2 alters the behavior of these nuclear foci and results in a decreased frequency of fusion events. We propose that SUMO modification is an important regulatory system for the mobility of the nuclear domains formed by Bach2.

Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging.

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)--BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)--for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.

Co-repressor SMRT and class II histone deacetylases promote Bach2 nuclear retention and formation of nuclear foci that are responsible for local transcriptional repression.

Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the TPA response element, the Maf recognition element (MARE) and the antioxidant-responsive element. Recently, it was reported that upon oxidative stress Bach2 forms nuclear foci surrounding the promyelocytic leukaemia (PML) bodies and specifically represses the transcription around the PML bodies. Here we report that expression of the silencing mediator of retinoid and thyroid receptor (SMRT) and histone deacetylase4 (HDAC4) enhances the formation of the Bach2 foci in the nuclear matrix. SMRT mediates the HDAC4 binding to Bach2, and HDAC4 facilitates the retention of Bach2 in the foci. Scratch transcription labelling and 3D-reconstruction from the confocal images demonstrated that transcription is suppressed in and around the Bach2 foci. Indeed, Bach2 bound MARE and repressed the expression from the chromosomally integrated MARE-driven reporter gene when co-expressed with SMRT and HDAC4. Our observations suggest that both SMRT and HDAC4 play an important role in nuclear retention and the Bach2 focus formation in the mammalian cell nucleus, which may contribute to the local transcription repression.

The transcriptional programme of antibody class switching involves the repressor Bach2.

Activated B cells differentiate to plasma cells to secrete IgM or, after undergoing class switch recombination (CSR), to secrete other classes of immunoglobulins. Diversification of antibody function by CSR is important for humoral immunity. However, it remains unclear how the decision for the bifurcation is made. Bach2 is a B-cell-specific transcription repressor interacting with the small Maf proteins whose expression is high only before the plasma cell stage. Here we show that Bach2 is critical for CSR and somatic hypermutation (SHM) of immunoglobulin genes. Genetic ablation of Bach2 in mice revealed that Bach2 was required for both T-cell-independent and T-cell-dependent IgG responses and SHM. When stimulated in vitro, Bach2-deficient B cells produced IgM, as did wild-type cells, and abundantly expressed Blimp-1 (refs 9, 10) and XBP-1 (ref. 11), critical regulators of the plasmacytic differentiation, indicating that Bach2 was not required for the plasmacytic differentiation itself. However, they failed to undergo efficient CSR. These findings define Bach2 as a key regulator of antibody response and provide an insight into the orchestration of CSR and SHM during plasma cell differentiation.

Repression of PML nuclear body-associated transcription by oxidative stress-activated Bach2.

Several lines of evidence suggest that gene expression is regulated not only by the interaction between transcription factors and DNA but also by the higher-order architecture of the cell nucleus. PML bodies are one of the most prominent nuclear substructures which have been implicated in transcription regulation during apoptosis and stress responses. Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. Bach2 forms nuclear foci associated with PML bodies upon oxidative stress. Here, we demonstrate that transcription activity associated with PML bodies is selectively repressed by the recruitment of Bach2 around PML bodies. Fluorescence recovery after photobleaching experiments revealed that Bach2 showed rapid turnover in the nuclear foci. The Bach2 N-terminal region including the BTB domain is essential for the focus formation. Sumoylation of Bach2 is required for the recruitment of the protein around PML bodies. These observations represent the first example of modulation of transcription activity associated with PML bodies by a sequence-specific transcription factor upon oxidative stress.

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene.

Heme oxygenase-1 (HO-1) protects cells from various insults including oxidative stress. Transcriptional activators, including the Nrf2/Maf heterodimer, have been the focus of studies on the inducible expression of ho-1. Here we show that a heme-binding factor, Bach1, is a critical physiological repressor of ho-1. Bach1 bound to the multiple Maf recognition elements (MAREs) of ho-1 enhancers with MafK in vitro and repressed their activity in vivo, while heme abrogated this repressor function of Bach1 by inhibiting its binding to the ho-1 enhancers. Gene targeting experiments in mice revealed that, in the absence of Bach1, ho-1 became expressed constitutively at high levels in various tissues under normal physiological conditions. By analyzing bach1/nrf2 compound-deficient mice, we documented antagonistic activities of Bach1 and Nrf2 in several tissues. Chromatin immunoprecipitation revealed that small Maf proteins participate in both repression and activation of ho-1. Thus, regulation of ho-1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor-activator antagonism.

Expression of the oxidative stress-regulated transcription factor bach2 in differentiating neuronal cells.

Bach2 is an oxidative stress-regulated transcription factor and functions as a repressor of gene expression directed by the TPA-response element, the Maf recognition element, and the antioxidant responsive element. To investigate the possibility that these enhancers are regulated in a tissue-specific manner, we analyzed expression of Bach2 during differentiation of neural cells. Bach2 was induced upon neuronal differentiation of P19 embryonic carcinoma cells, while its related factor Bach1 did not show significant change. By using affinity-purified anti-Bach2 antibodies, expression of Bach2 in mouse embryos was determined. High levels of Bach2 antigen were found in differentiating neuronal and lens cells in day 12.5 embryos. Consistent with the fact that subcellular localization of Bach2 is regulated by nuclear export in cultured cells, extensive Bach2-staining was found in the cytoplasmic regions of developing neuronal and lens cells. These results suggest that Bach2 regulates AP-1- and Maf-dependent gene expression during development of neuronal and lens cells and that its activity may be regulated by nuclear export in these cells.