Real-time monitoring of stromal NADPH levels in Arabidopsis using a metagenome-derived NADPH-binding fluorescent protein.

The light irradiation to the plant chloroplasts drives NADPH and ATP synthesis in the stroma via the electron transport chains within the thylakoid membranes. Conventional methods for assessing photosynthetic light reactions are often invasive or require specific conditions. While detection markers do not significantly affect plant growth itself, developing a method for the real-time and non-invasive detection of NADPH is a highly impactful and important research area in plant physiology and biochemistry.

Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo.

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation.

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications.

One-step metal affinity purification of recombinant hFGF19 without using tags.

Human fibroblast growth factor 19 (hFGF19) belongs to the endocrine FGF19 superfamily and is considered a potential agent to treat severe or relapsing nonalcoholic fatty liver disease. Numerous studies have confirmed the beneficial effects of this hormone on the related symptoms of the disease and attempts at producing recombinant proteins in various hosts are steadily proliferating. Recently, we reported that authentic hFGF19 can be solubly expressed through combining synonymous codon substitutions and co-expression with disulfide-bond isomerase (DsbC) in Escherichia coli.

A designed fusion tag for soluble expression and selective separation of extracellular domains of fibroblast growth factor receptors.

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy.

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA.

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic-hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step.

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in .

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein.

Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5'-coding region in Escherichia coli.

Objective: To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed.

Results: We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.

Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP.

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems.

Rapid and sensitive detection of NADPH via mBFP-mediated enhancement of its fluorescence.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately.

An Alternative Platform for Protein Expression Using an Innate Whole Expression Module from Metagenomic DNA.

Many integrated gene clusters beyond a single genetic element are commonly trapped as the result of promoter traps in (meta)genomic DNA libraries. Generally, a single element, which is mainly the promoter, is deduced from the resulting gene clusters and employed to construct a new expression vector. However, expression patterns of target proteins under the incorporated promoter are often inconsistent with those shown in clones harboring plasmids with gene clusters.

Conditional constitutive expression system of a drug protein in vivo by positive feedback loop using an inducer-independent artificial transcription factor.

Bacterial-mediated drug delivery is a potential and promising strategy for the specific treatment of cancer with therapeutic molecules, especially with genetically encoded proteins. These proteins must be tightly regulated due to cytotoxicity and thus are usually expressed under the control of the P and TetA/TetR promoters in vivo. Since protein expression from these systems is triggered by exogenous inducer, periodic intravenous injection of inducer is necessary.

Antimicrobial and Anti-Biofilm Activities of the Methanol Extracts of Medicinal Plants against Dental Pathogens and .

Several medicinal plants are ethnomedically used in Korea as agents for treating infection, anti-inflammation, and pain relief. However, beyond typical inhibitory effects on cell growth, little is known about the potential anti-biofilm activity of these herbs, which may help to prevent cavities and maintain good oral health. This study aimed to investigate the antimicrobial and anti-biofilm activities of the methanol extracts of 37 Korean medicinal plants against dental pathogens and , which synergize their virulence so as to induce the formation of plaque biofilms in the oral cavity.

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon-substituted 5'-coding region variant of the cadA gene.

Aspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli.

Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource.

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E.

Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region.

Recent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein.

Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system.

Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system.

Construction of a T-vector using an esterase reporter for direct cloning of PCR products.

We constructed an efficient T vector pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones due to insertional inactivation of the esterase reporter. Additionally, PCR products were cloned into this vector efficiently without the gel purification steps, due to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.

Optimization of electrotransformation conditions for Propionibacterium acnes.

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P.

A novel assay system for the measurement of transketolase activity using xylulokinase from Saccharomyces cerevisiae.

The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates.

Analyses of the structural organization of unidentified open reading frames from metagenome.

Although there is no sequence information, activity-based screening methods can select positive clones from a metagenomic library. However, the low frequency of positive hits that is caused by improper expression of proteins in the cloning host Escherichia coli might be improved. In order to investigate whether the metagenome can be expressed in E.