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. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.

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. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

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. However, during purification, hFGF19 without the His-tag occasionally co-eluted with His-tagged DsbC when using metal affinity chromatography, thereby requiring auxiliary purification steps to achieve apparent homogeneity. This phenomenon provides evidence that hFGF19 specifically interacts with immobilized Ni2+, which can thus be used as an alternative tool for the purification of hFGF19. Consequently, we could simply and reproducibly purify hFGF19 from cell lysates by using Ni2+-immobilized metal affinity chromatography and stepwise gradient elution with imidazole.

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. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.

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. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

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

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 hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

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 (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. CONCLUSION: Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).

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. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.

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. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.

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. These results suggest that the integrated set of gene clusters with diverse cis- and trans-acting elements is evolutionarily tuned as a complete set for gene expression, and is an expression module with all the components for the expression of a nested open reading frame (ORF). This possibility is further supported by truncation and/or serial deletion analysis of this module in which the expression of the nested ORF is highly fluctuated or reduced frequently, despite being supported by plentiful cis-acting elements in the spanning regions around the ORF such as the promoter, ribosome binding site (RBS), terminator, and 3'-/5'-UTRs for gene expression. Here, we examined whether an innate module with a naturally overexpressed gene could be considered as a scaffold for an expression system. For a proof-of-principle study, we mined a complete expression module with an innately overexpressed ORF in E. coli from a metagenomics DNA library, and incorporated it into a vector that had no regulatory element for expressing the insert. We obtained successful expression of several inserts such as MBP, GFPuv, ß-glucosidase, and esterase using this simple construct without tuning and codon optimization of the target insert.

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 PBAD and TetA/TetR promoters in vivo. Since protein expression from these systems is triggered by exogenous inducer, periodic intravenous injection of inducer is necessary. However, these treatments can result in non-homogenous and/or inefficient expression of therapeutic proteins in vivo due to impeded diffusion and dilution of the inducer further from the injection site. To overcome these hurdles, we designed a conditional constitutive expression system equipped with the artificial transcription factor, AraCC, which has two operator-binding domains and simultaneously binds to the I1 and I2 operators of the PBAD promoter for gene expression in an arabinose-independent manner. Using this construct and the wild type protein AraC under the control of the PBAD promoter, we constructed a self-positive feedback system to constitutively express the therapeutic protein when the induction of AraC was triggered once using arabinose. This expression system could be useful in various cancer treatment strategies using bacteria to deliver genetically encoded drugs in vivo.

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

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 Streptococcus mutans and Candida albicans, which synergize their virulence so as to induce the formation of plaque biofilms in the oral cavity. The antimicrobial activities were investigated by broth dilution and disk diffusion assay. The anti-biofilm and antioxidant activities were evaluated based on the inhibitory effect against glucosyltransferase (GTase) and the DPPH assay, respectively. Among 37 herbs, eight plant extracts presented growth and biofilm inhibitory activities against both etiologic bacteria. Among them, the methanol extracts (1.0 mg/ml) from Camellia japonica and Thuja orientalis significantly inhibited the growth of both bacteria by over 76% and over 83% in liquid media, respectively. Minimum inhibitory concentration (MIC) values of these methanol extracts were determined to be 0.5 mg/ml using a disk diffusion assay on solid agar media. Biofilm formation was inhibited by more than 92.4% and 98.0%, respectively, using the same concentration of each extract. The present results demonstrate that the medicinal plants C. japonica and T. orientalis are potentially useful as antimicrobial and anti-biofilm agents in preventing dental diseases.

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. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85 g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6 ± 33.4 mg/L IA during a 72-h culture after induction with isopropyl ß-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2 g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;113: 1504-1510. © 2015 Wiley Periodicals, Inc.

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. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

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. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.

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. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-ß-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10(4) clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.

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. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.

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. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 x 10(-4)unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.

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. coli, the structural organization of URFs from metagenome was analyzed in terms of transcription and translation factors, and compared to those of 4300 ORFs of E. coli K12. Considerable differences in amino acid composition and codon usage occurred between the metagenome URFs and E. coli ORFs, reflecting a barrier for protein expression within the host E. coli. From the analyses of the promoter and RBS regions, sequences or patterns in the corresponding region of metagenome URFs were found to be dissimilar to E. coli consensus. These results suggested that these factors are considerable to screen the clones from metagenomic library with the activity-based approach.