Biomedical subjects

Enoch Y Park

Publications and source records attributed to Enoch Y Park.

At least 19 recordsLinked to original sources

PubMed2006

Construction of hybrib Autographa californica nuclear polyhedrosis bacmid by modification of p143 helicase.

We developed a new hybrid nuclear polyhedrosis virus (NPV) bacmid capable of infecting Spodoptera frugiperda, Tricoplusia ni, and Bombyx mori, and B. mori cell lines for producing hybrid recombinant baculovirus that can carry a gene of interest and express it in a broad range of hosts. A GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 fusion gene was expressed successfully in silkworm larvae using this hybrid bacmid. The hybrid NPV bacmid provides an altogether simple and realistically feasible method for large-scale applications using silkworm larvae. It can be easily managed in E. coli, which has no biohazard safety concerns, in addition to the baculovirus-based expression system.

Animals↗

PubMed2006

Identification of epitope on DNA-binding protein expressed in insect cell infected by baculovirus.

DNA-binding protein (DBP) is an early gene product produced during viral replication. Polyclonal anti-DBP was produced using rabbit by intradermal injections of Escherichia coli-expressed purified recombinant DBP. Prepared anti-DBP completely blocked the replication of baculovirus in insect cells. The anti-DBP binding to DBP was confirmed by both Western blotting with Tn-5B1-4 insect cell lysates as well as immunostained baculovirus-infected Tn-5B1-4 insect cells. To determine the anti-DBP epitope 12 peptides were synthesized and their specific-binding activities were measured using ELISA. Based on specific-binding activity against anti-DBP the epitope was predicted to be between amino acid residues 248-265 (QRMSVEDFDRLFEMDKID). Especially from 18 amino acid residues it was further to be narrowed between amino acid residues 260-265 (EMDKID) which showed a critical role in specific-binding activity.

Animals↗

PubMed2006

Multiple co-transfection and co-expression of human beta-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells.

Human beta-1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, beta3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25-1500 microg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed beta3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying beta3GnT2. In this study we correlated the activity of beta3GnT2 with the amount of beta3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and beta3GnT2 cDNA were in a rough ratio of 1:1, the beta3GnT2 activity was at a high level. In order to achieve better expression levels of beta3GnT2 with less cost and time, efficient ways have to be devised.

Animals↗

PubMed2006

Binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system.

The rat recombinant renin/prorenin receptor (AB188298 in DDJB), which conjugated with FLAG epitope in its N-terminus, was expressed in a baculovirus expression system. The recombinant receptor, prepared from the cytoplasmic fraction of the insect cells, was identified by Western blotting using anti-FLAG antibody. Prorenin as well as renin bound to the receptor with different binding affinities. Their Kd values were estimated at 8.0 and 20 nM, respectively. The amounts of prorenin and renin bound to the immobilized receptors were 1.0 and 0.2 pmole, respectively. The prorenin bound to the receptor had renin activity and the renin kept the activity at similar level to that before the binding. The Km of their complexes was the same at 3.3 microM when sheep angiotensinogen was used as the substrate. Their Vmax values were 5.5 and 10 nM.h(-1), respectively. The molecular activities of prorenin and renin bound to the receptor were 1.1 and 10 h(-1), respectively. From these findings, rat prorenin as well as renin was indicated to bind to the recombinant receptor and express the enzymatic activity in vitro.

Animals↗

PubMed2005

Expression of alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae in Ashbya gossypii.

Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 alpha (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10-20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg-1 of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l-1, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.

Alanine Transaminase↗

PubMed2005

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Amino Acid Sequence↗

PubMed2005

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion.

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.

Amino Acid Sequence↗

PubMed2005

Improvement of the production of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system.

A stable Tn-5B1-4 insect cell line co-expressing the recombinant GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 (GFPuv-beta3GnT2) protein fused to a melittin signal sequence with a lectin-like molecular chaperone, human calnexin (hCNX) or human calreticulin (hCRT), was constructed. The expression of either of these molecular chaperones is under the control of a weak promoter, OpMNPV IE2, while that of GFPuv-beta3GnT2 is under the control of Bombyx mori actin promoter. This co-expression system was compared between two different insect cell-baculovirus expression systems: (1) co-infection of the recombinant baculovirus containing a molecular chaperone (AcNPV-hCNX or -hCRT) with a recombinant baculovirus containing GFPuv-beta3GnT2 fused with the melittin signal sequence (AcNPV-me-GGT); (2) infection of AcNPV-me-GGT to a stably expressing cell line for either hCNX or hCRT. In the co-infection system, the intracellular GFPuv-beta3GnT2 expression level was low because of the improved secretion level ratio of the fusion protein, due to the chaperone expression. In the case of infection to the stably expressing cell line for a chaperone, the extracellular GFPuv-beta3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process beta3GnT2. On the other hand, the co-expression system produced an extracellular beta3GnT activity of 22-23 mU/mL, which was approximately 3.5- and 11-fold higher than those of the stable expression of the fusion gene without the chaperone and the conventional BES with the addition of protease, respectively. The secretion level ratio of the fusion protein of this system increased to 82%, which was approximately 1.5-fold that of any other expression system investigated thus far. These results indicate that the ratio of the expression level of the target gene to that of the chaperone gene may be an important factor in maximizing the production of a target protein. The molecular-chaperone-assisted expression system using a stably transformed insect cell line offers promising prospects for the efficient production of recombinant secretory proteins in insect cells.

Animals↗

PubMed2005

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.

Animals↗

PubMed2005

Use of plant-derived protein hydrolysates for enhancing growth of Bombyx mori (silkworm) insect cells in suspension culture.

The successful suspension culture of the Bombyx mori (silkworm) cell lines Bm5 and BmN4 without FBS (fetal-bovine serum) was first realized in Sf-900 II SFM (serum-free medium) (Gibco BRL, Rockville, MD, U.S.A.) supplemented with a plant-derived protein hydrolysate. The addition of 0.5% HyPep 1510 (Difco Co., Detroit, MI, U.S.A.) and 10 mM glutamine to Sf-900 II SFM at 4 days of culture was found effective in increasing the cell concentration to 8.5x10(6) cells/ml. The replacement of medium with Sf-900 II SFM supplemented with 0.5% HyPep 1510 at 6 days of culture increased the cell concentration by 1.1x10(7) cells/ml. When Sf-900 II SFM was supplemented with 0.5% Hypep 1510, 16 days, which was half of the conventional adaptation time, was sufficient for the B. mori cell line to adapt to SFM and shear stress while maintaining a stable viability. The beta-galactosidase activity in Sf-900 II SFM supplemented with 0.5% Hypep 1510 was 4.9x10(3) units/ml, which was 2-fold higher than that of the FBS-supplemented medium. By SDS/PAGE, only the band corresponding to beta-galactosidase was detected in the sample from the media supplemented with plant-derived protein hydrolysates, while thick bands corresponding to proteins having lower molecular masses than beta-galactosidase were detected in samples from the FBS-supplemented media. These results suggest that plant-derived protein hydrolysates are promising FBS substitutes for enhancing the growth of B. mori cells and facilitating the purification of recombinant proteins produced by baculovirus infection.

Adaptation, Physiological↗

PubMed2005

Application of a radial-flow bioreactor in the production of beta1,3-N-acetylglucosaminyltransferase-2 fused with GFPuv using stably transformed insect cell lines.

An RFB (radial-flow bioreactor) with a reactor volume of 5 ml was applied to produce human beta3GnT (beta1,3-N-acetylglucosaminyltransferase) using two stably transformed insect cell lines. When air was supplied to the RFB, cell growth stopped at 4 days of culture and beta3GnT was not detected. However, with a supply of pure oxygen, the cell concentration, assumed from glucose consumption, increased by 1.3x10(7) cells/ml. Insect cells attached to poly(vinyl alcohol) matrixes packed in the RFB and grew confluently; 5.6 m-units/ml beta3GnT was produced under the conditions of pure oxygen supply and addition of glucose and glutamine. This RFB was first applied in beta3GnT production using stably transformed insect cells. The amount of beta3GnT production in only a 5 ml-scale RFB was comparable with that of a 100 ml shaking flask culture.

Animals↗

PubMed2004

Recent progress in microbial cultivation techniques.

Recent advances in the improvement of microbial cultivation are reviewed, with emphasis on biochemical engineering techniques as a means of obtaining high production rate of bioproduct. Possible uses of high cell density culture include their use in food industry as well as in the production of new medicines and in biotechnology. Concentration of microorganisms using a hollow fiber membrane or centrifuge, and increase in cell density by controlling the pH, dissolved oxygen, or carbon source concentrations of the culture broth with control algorithms are discussed. In a culture of filamentous microorganisms the mycelial morphology is hard to define and it is difficult to quantify its amount, and this is one of the bottlenecks hampering the improvement of production rate. Specific features of mycelial cultivation in the presence of highly pulpy mycelia and entangled-pellets are scrutinized by visual inspection through a microscope that is linked to a computer, and using software that can characterize the mycelial morphology. Image analysis technology for analyzing the mycelial image captured by a digital camera is a potential tool for morphological analysis, including analysis of the morphological development of filamentous microorganisms.

Algorithms↗

PubMed2004

Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae.

L(+)-lactic acid production was investigated using an enzymatic hydrolysate of waste office automation (OA) paper in a culture of the filamentous fungus Rhizopus oryzae. In 4 d culture, 82.8 g/l glucose, 7 g/l xylose, and 3.4 g/l cellobiose contained in the hydrolysate were consumed to produce 49.1 g/l of lactic acid. The lactic acid yield and production rate were only 0.59 g/g and 16.3 g/l/d, respectively, only 75% and 61% of the results from the glucose medium. The low production rate from waste OA hydrolysate was elucidated by trials using xylose as the sole carbon source; in those trials, the lactic acid production rate was 7.3 g/l/d, only 28% that of glucose or cellobiose. The low lactic acid yield from waste OA hydrolysate was clarified by trials using artificial hydrolysates comprised of 7:2:1 or 7:1:2 ratios of glucose:cellobiose:xylose. For both, the lactic acid production rate of 17.4 g/l/d matched that of waste OA paper, while the lactic acid yield was similar to that of the glucose medium. This indicates that the production rate may be inhibited by xylose derived from hemicellulose, and the yield may be inhibited by unknown compounds derived from paper pulp.

Acremonium↗

PubMed2004

Comparative analysis of GFP(UV)-beta1,3-N-acetylglucosaminyltransferase 2 production in two insect-cell-based expression systems.

Active beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) was produced in the baculovirus expression system (BES) and in stably transformed insect Tn-5B1-4 cells. beta3GnT2 was expressed as a secreted fusion protein with GFP(UV) with three different types of signal sequence to enhance the secretion of the fusion protein. In the stably transformed cells, the maximal beta3GnT2 activity differed between isolates, but their secretion efficiencies were similar. The difference between the maximal beta3GnT activities of the isolates studied was considered to be due to the presence of a copy number of the fusion gene, as determined on the basis of the results of Southern blot analysis. The beta3GnT activities of the culture supernatant in BES (Tn-5B1-4 cells) without or with the addition of the protease inhibitor, leupeptin, were 0.68 and 2.01 mU/ml, respectively. The stably transformed Tn-5B1-4 cells (Tn-pXme11) exhibited a beta3GnT activity of 6.83 mU/ml, which was 3.4-fold higher than that observed for BES with the leupeptin addition. The purity of fusion protein purified from the culture supernatant of the Tn-pXme11 was higher than 95% on SDS-PAGE, in contrast with that purified from the culture supernatant of the baculovirus-infected cells which contained low-molecular-weight fragments of the fusion protein. The stably transformed cell line is more suitable than BES for the efficient production of the secretory protein, beta3GnT2.

Animals↗

PubMed2004

Oxidation of rapeseed oil in waste activated bleaching earth and its effect on riboflavin production in culture of Ashbya gossypii.

Long-term behavior of rapeseed oil in waste activated bleaching earth (ABE) and the effect of this oil on riboflavin production in the culture of Ashbya gossypii were investigated. Waste ABE with 40% (w/w) rapeseed oil was stored for 80 d, and the extent of oxidation of rapeseed oil was measured by several analytical methods to determine the chemical properties of the oil at different stages of the oil deterioration process:peroxide value, acid value, concentrations of organic acids, acetaldehyde and unsaturated fatty acid, and content of polymerized triglycerides. Peroxide value, acid value, and concentrations of organic acids and acetaldehyde did not affect riboflavin production. However, the content of polymerized triglycerides markedly increased the viscosity of rapeseed oil and was the main reason for the exponential decrease in riboflavin production. A good correlation between the polymerized triglyceride content or viscosity and riboflavin production in the culture of A. gossypii using rapeseed oil as the sole carbon source was found.

Journal Article↗

PubMed2004

Efficient production of fatty acid methyl ester from waste activated bleaching earth using diesel oil as organic solvent.

Fatty acid methyl ester (FAME) production from waste activated bleaching earth (ABE) discarded by the crude oil refining industry was investigated using fossil fuel as a solvent in the esterification of triglycerides. Lipase from Candida cylindracea showed the highest stability in diesel oil. Using diesel oil as a solvent, 3 h was sufficient to obtain a yield of approximately 100% of FAME in the presence of 10% lipase from waste ABE. Kerosene was also a good solvent in the esterification of triglycerides embedded in the waste ABE. Fuel analysis showed that the FAME produced using diesel oil as a solvent complied with the Japanese diesel standard and the 10% residual carbon amount was lower than that of FAME produced using other solvents. Use of diesel oil as solvent in the FAME production from the waste ABE simplified the process, because there was no need to separate the organic solvent from the FAME-solvent mixture. These results demonstrate a promising reutilization method for the production of FAME, for use as a biodiesel, from industrial waste resources containing waste vegetable oils.

Journal Article↗

PubMed2003

Improvement of GFPuv-beta 3GnT2 fusion protein production by suppressing protease in baculovirus expression system.

The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 microg/ml improved intra- and extracellular beta 3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of beta 3GnT production, judging from SDS-PAGE. Moreover, when 50 microM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular beta 3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein.

Animals↗

PubMed2002

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G(2)/M phase with 4n DNA. In the case of G(1) or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G(2)/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G(1) phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G(2)/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G(1) cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G(1) or S phase-infection was 1.5-1.8-fold higher than that at G(2)/M phase-infection.

Animals↗