Joon-Ki Jung

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

Haemophilus parasuis causes contagious porcine Glässers 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.

Enhancement of monascus pigment production by the culture of Monascus sp. J101 at low temperature

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 °C. In a batch culture at 30 °C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 °C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 °C was 10 times greater than at 30 °C.

Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter

ABSTRACT 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 (PPHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. PPHO89 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 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, PPHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple PPHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of PPHO89 for controlled production of recombinant proteins in P. pastoris.

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.

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 S3N10 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 nmolCBD-GAD/gAvicel 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.

Highly efficient production of monoglycerides by the continuous removal of fatty acids from lipase-catalyzed oil hydrolysis

Highly efficient production of monoglycerides was achieved from lipase-catalyzed oil hydrolysis by the continuous addition of CaCl2 to remove the fatty acids produced. A fusion protein produced by connecting a cellulose-binding domain of Trichoderma hazianum cellulase to Bacillus stearothermophilus L1 lipase was used as a model 1,3-regiospecific lipase. The reaction was performed at pH 10 and 50°C, and the relationship between continuous removal of fatty acids and the production of monoglyceride was investigated by microscopic and HPLC analysis of oil emulsions and the reaction products. Without the addition of Ca2 + the reaction was inhibited by fatty acids, with the decrease in reaction rate being proportional to the concentration of fatty acids. When CaCl2 was continuously added in a 1:2 molar ratio with the released fatty acids, the reaction progressed unimpeded due to the formation of Ca-soaps. Both the yield and the fraction of monoglyceride in the reaction product increased due to the continuous removal of fatty acids.

Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae: Effect of different feeding strategies

Out of four different feeding strategies tested for the production of extracellular recombinant glucose oxidase from Saccharomyces cerevisiae, constant feeding of galactose on the exhaustion of initial glucose, gave the highest yield-154 U/ml which was 62% above the yield achieved in batch operation (95 U/ml).

Effects of Mixing on Fed-Batch Fermentation of L-Ornithine

The effects of mixing on L-ornithine fermentation were studied using an L-arginine auxotrophic mutant of Brevibacterium ketoglutamicum ATCC 21092. Three different modes of fed-batch culture using two different impeller types were studied in a 7 l fermentor. The first two modes of fed-batch culture were carried out in a fermentor equipped with six-flat-blade disk-turbine impellers with top and bottom feeding. The third mode of fed batch culture was carried out using pitched-six-flat-blade disk-turbine impellers with top feeding. The titre of L-ornithine increased up to 1.8 fold with bottom-feeding or when the pitched-six-flat-blade disk-turbine impellers were used compared to when the six-flat-blade disk-turbine impellers with top-feeding were used. It was observed that the mixing time of the limiting nutrients varied significantly depending on both the direction of feeding and the impeller type. Since the profiles of the specific rates of CO2 evolution, oxygen and glucose uptakes were very similar for the three culture modes, it could be reasonably assumed that the microorganism exhibited similar growth rates for each mode used. However, different amino acid producing activities were observed in the three culture modes. From these results it is concluded that the productivity of L-ornithine fermentation is significantly improved by shortening the mixing time of the limiting nutrient in the fermentor.

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.

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

BackgroundThe 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.ResultsAn 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 (qAb) than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones.ConclusionsThis study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb 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.