Increased genomic integrity of an improved protein-based mouse induced pluripotent stem cell method compared with current viral-induced strategies.

It has recently been shown that genomic integrity (with respect to copy number variants [CNVs]) is compromised in human induced pluripotent stem cells (iPSCs) generated by viral-based ectopic expression of specific transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc). However, it is unclear how different methods for iPSC generation compare with one another with respect to CNV formation. Because array-based methods remain the gold standard for detecting unbalanced structural variants (i.e., CNVs), we have used this approach to comprehensively identify CNVs in iPSC as a proxy for determining whether our modified protein-based method minimizes genomic instability compared with retro- and lentiviral methods. In this study, we established an improved method for protein reprogramming by using partially purified reprogramming proteins, resulting in more efficient generation of iPSCs from C57/BL6J mouse hepatocytes than using protein extracts. We also developed a robust and unbiased 1 M custom array CGH platform to identify novel CNVs and previously described hot spots for CNV formation, allowing us to detect CNVs down to the size of 1.9 kb. The genomic integrity of these protein-based mouse iPSCs (p-miPSCs) was compared with miPSCs developed from viral-based strategies (i.e., retroviral: retro-miPSCs or lentiviral: lenti-miPSCs). We identified an increased CNV content in lenti-miPSCs and retro-miPSCs (29∼53 CNVs) compared with p-miPSCs (9∼10 CNVs), indicating that our improved protein-based reprogramming method maintains genomic integrity better than current viral reprogramming methods. Thus, our study, for the first time to our knowledge, demonstrates that reprogramming methods significantly influence the genomic integrity of resulting iPSCs.

Isolation and characterization of a novel ε-caprolactam-degrading microbe, Acinetobacter calcoaceticus, from industrial wastewater by chemostat-enrichment.

For the isolation of a ε-caprolactam-degrading microbe from wastewaters of a factory producing caprolactam, we applied a chemostat-enrichment technique with a selective medium containing caprolactam as sole source of carbon and nitrogen. This allowed for the isolation of a novel caprolactam-degrading microbe, identified as Acinetobacter calcoaceticus. The strain had a critical tolerance of 19 g caprolactam l(-1) in minimal medium, which is higher than any previously reported caprolactam-degrading microbe. A. calcoaceticus also decreased the caprolactam content in medium by 65 % within 72 h despite the high caprolactam content (10 g l(-1)). This study highlights the potential use of A. calcoaceticus strain for the bioremediation of recalcitrant synthetic polymers, such as caprolactam.

Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography.

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.

Soluble expression of OmpA from Haemophilus parasuis in Escherichia coli and its protective effects in the mouse model of infection.

Haemophilus parasuis causes contagious porcine Glässer's disease leading to severe losses in the swine industry. In this study, we established an efficient Escherichia colibased system for the expression of H. parasuis major outer-membrane protein (MOMP) that has been known as a good vaccine candidate against Glässer's disease. Use of an E. coli-derived pelB leader sequence made it possible to produce recombinant MOMP (rMOMP) as the soluble forms without an additional refolding process. Using two different animal models, it was evaluated that the rMOMP was capable of inducing a significant immune response and providing protection against H. parasuis infection.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BACKGROUND: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. RESULTS: An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. CONCLUSIONS: This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

Expression, immobilization and enzymatic properties of glutamate decarboxylase fused to a cellulose-binding domain.

Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamic acid to gamma-aminobutyric acid (GABA), was fused to the cellulose-binding domain (CBD) and a linker of Trichoderma harzianum endoglucanase II. To prevent proteolysis of the fusion protein, the native linker was replaced with a S(3)N(10) peptide known to be completely resistant to E. coli endopeptidase. The CBD-GAD expressed in E. coli was successfully immobilized on Avicel, a crystalline cellulose, with binding capacity of 33 ± 2 nmol(CBD-GAD)/g(Avicel) and the immobilized enzymes retained 60% of their initial activities after 10 uses. The results of this report provide a feasible alternative to produce GABA using immobilized GAD through fusion to CBD.

NADPH-dependent pgi-gene knockout Escherichia coli metabolism producing shikimate on different carbon sources.

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+) strain and its mixed glucose/fructose fermentation grew well. Interestingly, when fructose was used as a carbon source, the pgi(-) mutant showed the enhanced SA production compared with the pgi(+) strain. In silico analysis of a genome-scale E. coli model was then conducted to characterize the cellular metabolism and quantify NAPDH regeneration, which allowed us to understand such experimentally observed attenuated cell growth and enhanced SA production in glucose- and fructose-consuming pgi(-) mutant, respectively with respect to cofactor regeneration.

Carbon nanotube-assisted enhancement of surface plasmon resonance signal.

We describe a method of amplifying the biosensing signal in surface plasmon resonance (SPR)-based immunoassays using an antibody-carbon nanotube (CNT) conjugate. As a model system, human erythropoietin (EPO) and human granulocyte macrophage colony-stimulating factor (GM-CSF) were detected by sandwich-type immunoassays using an SPR biosensor. For the amplification of the SPR signal, the CNT was conjugated with a polyclonal antibody, and then the conjugates were reacted with antibodies coupled with the target proteins. This amplification strategy increases the dynamic range of the immunoassays and enhances the detection sensitivity. The SPR immunoassays, combined with the CNT-assisted signal amplification method, provided a wide dynamic range over four orders of magnitude for both EPO and GM-CSF (0.1-1,000 ng/ml). The CNT amplification method is expected to realize the detection of picogram levels and a wide dynamic detection range of multiple proteins, enabling it to offer a robust analysis tool for the development of biopharmaceutical production.

Identification of novel immunogenic proteins in pathogenic Haemophilus parasuis based on genome sequence analysis.

Haemophilus parasuis causes contagious porcine Glässer's disease, which is occurring worldwide and leads to severe losses in the pig industry. To identify novel antigen candidates against this disease, 22 surface-exposed or secreted proteins were selected from the annotated H. parasuis genome by reverse vaccinology strategy. Expression of these proteins in Escherichia coli was attempted. Immunogenicity of the expressed candidates was assessed using Western blot analysis with mouse-derived antiserum prepared with whole bacteria of H. parasuis serovar 4 or 5. Three ABC-type transporters (OppA, YfeA and PlpA) and 1 curli protein assembly (CsgG) were identified as potent immunogenic proteins. The proteins show cross-reactions when tested with sera raised against serovars 4 and 5 of H. parasuis.

Osteopontin splice variants differentially modulate the migratory activity of hepatocellular carcinoma cell lines.

Osteopontin (OPN, SPP1) is a secretory extracellular matrix protein that has been implicated in cancer-associated mechanisms such as metastasis, invasion and angiogenesis. Three OPN isoforms (OPN-a, -b and -c) derived from alternative splicing are known to exist, but their functional specificity remains unclear. Here, we found that the expression profile of OPN isoforms in hepatocellular carcinoma (HCC) cell lines and patient tissues were correlated with specific cellular phenotypes and tumorigenicity of HCC. Thus, SK-Hep1 cells with a robust migratory capacity dominantly expressed both OPN-a and -b, but non-migratory cell lines such as Hep3B and PLC/PRF/5 mainly expressed OPN-c. Moreover, tumor tissues predominantly expressed OPN-a and -b, whereas normal liver tissues mainly expressed OPN-c. Transwell infiltration and wound-induced migration assays revealed that both OPN-a and -b induced Hep3B cell migration, while OPN-c had no significant effects. By contrast, OPN-c suppressed the migratory activity of SK-Hep1 cells although no significant changes were induced by OPN-a. Consistently, OPN isoforms differentially activated migration-associated signaling pathways such that OPN-a and -b increased the expression of urokinase type plasminogen activator and the phosphorylation of p42/p44 MAP kinase, but these pathways were not activated by OPN-c. Thus, the findings of the present study suggest that OPN splice variants differentially couple to signaling pathways to modulate the migratory property of HCC cells and that this is one of the mechanisms underlying the pathological heterogeneity of HCC progression.

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.

Exploring the effects of carbon sources on the metabolic capacity for shikimic acid production in Escherichia coli using in silico metabolic predictions.

Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli was conducted to quantify the theoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yield and energy utilization, thereby identifying the indispensable pathways for achieving optimal SA production on each carbon source. Subsequently, a shikimate-kinase-deficient E. coli mutant was developed by blocking the aromatic amino acid pathway, and the production of SA on various carbon sources was experimentally examined during 5 l batch culture. As a result, the highest production rate, 1.92 mmol SA/h, was obtained when glucose was utilized as a carbon source, whereas the efficient SA production from glycerol was obtained with the highest yield, 0.21 mol SA formed per mol carbon atom of carbon source consumed. The current strain can be further improved to satisfy the theoretically achievable SA production that was predicted by in silico analysis.

Identification of an alternative translation initiation site for the Pantoea ananatis lycopene cyclase (crtY) gene in E. coli and its evolutionary conservation.

Previous sequence analyses of the lycopene cyclase gene (crt Y) from Pantoea ananatis revealed that translation of its protein product in Escherichia coli began at the ATG start codon. We found, however, that this enzyme could also be produced in E. coli without the ATG start codon present. Results of experiments using crt Y mutants revealed that a GTG (Val) sequence, located in-frame and 24 bp downstream of the ATG, could act as a potential start codon. Additionally, a point-mutated GTA (Val), replaced from alternative GTG start codon, also displayed its potential as a start codon although the strength as a translation initiation codon was considerably weak. This finding suggests that non-ATG codons, especially one base pairing with the anticodon (3'-UAC-5') in fMet-tRNA, might be also able to function as start codon in translation process. Furthermore, amino acid sequence alignment of lycopene cyclases from different sources suggested that a Val residue located within the N-terminus of these enzymes might be used as an alternative translation initiation site. In particular, presence of a conserved Asp, located in-frame and 12 bp upstream of potential start codon, supports this assumption in view of the fact that Asp (GAT or GAC) can function as part of the Shine-Dalgano sequence (AGGAGG).

Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides.

Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3.IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3.IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3.IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3.IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3.IL-2/HE molecule. The G3.IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16).

Identification of a fibrinolytic enzyme by Bacillus vallismortis and its potential as a bacteriolytic enzyme against Streptococcus mutans.

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg(-1). Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.

Over-production of beta-carotene from metabolically engineered Escherichia coli.

To produce recombinant beta-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. beta-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient beta-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal beta-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l(-1) and 240 mg l(-1), respectively, with overall productivities of 7.8 mg l(-1) h(-1) and 4.8 mg l(-1) h(-1).

Improved L-threonine production of Escherichia coli mutant by optimization of culture conditions.

L-threonine production was investigated in a minimal salt medium using L-threonine-overproducing Escherichia coli MT201, derived from E. coli K-12. It was observed that dry cell weight reached 12.5 g/l with 15.9 g/lL-threonine. To increase dry cell weight and L-threonine production, the fermentation process was optimized. When biotin was added as growth factor, L-threonine production reached 52.0 g/l from 15.9 g/l without biotin. Dry cell weight and L-threonine production were further increased by continuous feeding of the feed media with an optimized L-methionine concentration (5.0 g/l). However, high-cell-density culture caused oxygen-limited condition, which resulted in the accumulation of organic acids. To overcome this problem, oxygen-enriched air was supplied to the fermentor with the minimal salt medium. Under these optimal conditions, we achieved an L-threonine production of 80.2 g/l in the minimal salt medium.

Immuno-stimulating effect of the endo-polysaccharide produced by submerged culture of Inonotus obliquus.

Inonotus obliquus BELYU1102 was selected from 12 different strains of Inonotus as a producer of immuno-stimulating polysaccharide. After a batch fermentation of I. obliquus BELYU1102 was carried out in a 300 l pilot vessel, endo-polysaccharide and exo-polysaccharide were both obtained. The proliferation activity of endo-polysaccharide for splenic cells was much higher than the activity of exo-polysaccharide. The active endo-polysaccharide was produced primarily during the late stationary phase. Enhanced proliferation and polyclonal IgM antibody production were observed in B cells by purified water-soluble endo-polysaccharide. Nitrite production and expression of IL-1beta, IL-6, TNF-alpha, and iNOS in macrophages were also enhanced. However, the endo-polysaccharide did not affect the proliferation of T cells, the IL-2 expression of Th1 cells, or the IL-4 expression of Th2 cells. The endo-polysaccharide showed activities similar to lipopolysaccharide (LPS) for B cells and macrophages, but there was a large difference between the two polysaccharides because cellular activations induced by endo-polysaccharide were not affected by polymyxin B, a specific inhibitor of LPS. The endo-polysaccharide appeared to have other cellular binding sites with TLR-4 and did not show a direct toxicity against tumor cells. However, indirect anti-cancer effects via immuno-stimulation were observed. The mycelial endo-polysaccharide of I. obliquus is a candidate for use as an immune response modifier. Submerged mycelial cultures are advantageous for industrial production of polysaccharides.

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.