• Korean Journal of Microbiology
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• v.33 no.1
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• pp.1-6
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• 1997

5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.

• The Korean Journal of Mycology
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• v.27 no.1 s.88
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• pp.76-81
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• 1999

Exo-polysaccharide obtained from the submerged cultivation of Ganoderma lucidum mycelium was fractionated. The structural analysis of the acidic exo-polysaccharide fraction (BWS-DA-GI), showing high antitumor activity, was carried out and compared to the mycelial acidic fraction (MWS-DA-GI). The major sugar constituents of the fraction of BWS-DA-GI were glucose, galactose and mannose in the molar ratio of 2.5 : 2.1 : 2.5. The minor components in this fraction were xylose and fucose. While the major sugar constituents of the mycelial acidic fraction of MWS-DA-GI were galactose, fucose, mannose and glucose. The trace components in this fraction was xylose. From the results of periodate oxidation, Smith degradation, affinity chromatography and methylation analysis, the chemical structures of the two fractions, BWS-DA-GI and MWS-DA-GI were both determined as ${\beta}-1,3$ glucans. It was also estimated that BWS-DA-GI had a $1{\rightarrow}6$ glucosidic linkage and MWS DA-GI had $1{\rightarrow}4$ and $1{\rightarrow}6$ glucosidic linkages. The molecular weights of these fractions, MWS-DA-GI and MWSDA-GI were estimated as $1.2{\times}10^6\;and\;1.0{\times}10^6$ dalton, respectively.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.95-95
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• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.195-201
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• 2010

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1386-1392
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• 2008

A gene (sll0158) putatively encoding a glycogen branching enzyme (GBE, E.C. 2.4.1.18) was cloned from Synechocystis sp. PCC6803, and the recombinant protein expressed and characterized. The PCR-amplified putative GBE gene was ligated into a pET-21a plasmid vector harboring a T7 promoter, and the recombinant DNA transformed into a host cell, E. coli BL21(DE3). The IPTG-induced enzymes were then extracted and purified using Ni-NTA affinity chromatography. The putative GBE gene was found to be composed of 2,310 nucleotides and encoded 770 amino acids, corresponding to approx. 90.7 kDa, as confirmed by SDS-PAGE and MALDI-TOF-MS analyses. The optimal conditions for GBE activity were investigated by measuring the absorbance change in iodine affinity, and shown to be pH 8.0 and $30^{\circ}C$ in a 50 mM glycine-NaOH buffer. The action pattern of the GBE on amylose, an $\alpha$-(1,4)-linked linear glucan, was analyzed using high-performance anion-exchange chromatography (HPAEC) after isoamylolysis. As a result, the GBE displayed $\alpha$-glucosyl transferring activity by cleaving the $\alpha$-(1,4)-linkages and transferring the cleaved maltoglycosyl moiety to form new $\alpha$-(1,6)-branch linkages. A time-course study of the GBE reaction was carried out with biosynthetic amylose (BSAM; $M_p{\cong}$8,000), and the changes in the branch-chain length distribution were evaluated. When increasing the reaction time up to 48 h, the weight- and number-average DP ($DP_w$ and $DP_n$) decreased from 19.6 to 8.7 and from 17.6 to 7.8, respectively. The molecular size ($M_p$, peak $M_w{\cong}2.45-2.75{\times}10^5$) of the GBE-reacted product from BSAM reached the size of amylose (AM) in botanical starch, yet the product was highly soluble and stable in water, unlike AM molecules. Thus, GBE-generated products can provide new food and non-food applications, owing to their unique physical properties.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.49-54
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• 1995

To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolated cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and those of five clones containing poly(A) tail were compared with sequences of other plant viruses. One of these clones, V9, has a primary structure similar to the carlavirus group, suggesting that the clone V9 derived from a part of garlic latent virus (GLV). Northern blot analysis with the clone V9 as a probe demonstrated that GLV genome is 8.5 knt long and has a poly(A) tail. The clone V9 encodes coat protein (CP) of 33 kDa and nucleic acid binding protein of 10 kDa in different reading frame. The hexanucleotide motif, 5'-ACCUAA, which is conserved in the 3' noncoding region arid was proposed to be a cis-acting element involved in the production of negative strand genomic RNA was noticed. Complementary sequence to the hexanucleotide motif, 5'-TTAGGT, is also found in the positive strand of V9 RNA. The putative CP gene was cloned into the pRSET-A expression vector and expressed in E. coli BL21. The expressed recombinant V9CP protein was purified by $Ni^{2+}$ NTA affinity chromatography. The anti-V9CP antibody recognizes 34 kDa polypeptide which could be CP of GLV in infected garlic leaf extract. Immunoblot and Northern blot analysis of various cultivars shows wide occurrence of GLV in Korean garlic plants.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.261-268
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• 1998

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin. TPCK and TLCK, and also by $Hg^{2+}$, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.

• Applied Biological Chemistry
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• v.43 no.2
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• pp.100-105
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• 2000

Anionic peroxidases (POD) were isolated from Korean radish (Raphanus sativus L.) root by using fractionation with $(NH_4)_2SO_4$ and CM-cellulose ion exchange chromatography and used as the colorimetric enzyme for glucose determination. The chromogen used in this work was o-tolidine or 4-aminoantipyrine/diethylaniline (4AA/DEA) and the colored products were measured at 630 nm. Korean radish anionic POD showed much better colorimetric reaction of glucose determination with 4AA/DEA than with o-tolidine. The r values of calibration curve for glucose determination by o-tolidine and 4AA/DEA were 0.9983 and 0.9963, respectively. In order to compare the reactivity for substrate oxidation by Korean radish POD and horseradish POD, the Km values against o-dianisidine and guaiacol were measured. Korean radish POD had about 40 fold higher affinity for o-dianisidine and 2 fold higher affinity for guaiacol as revealed by Km values. These results showed that Korean radish POD could be developed as the colorimetric diagnosis reagent for glucose determination with high sensitivity.

• The Korean Journal of Food And Nutrition
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• v.23 no.2
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• pp.171-177
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• 2010

The catabolic threonine dehydratase from Serratia marcescens ATCC 25419 was purified to homogeniety using Sephadex G-200 gel filtration and AMP-Sepharose 4B affinity chromatography. The molecular weight of the native enzyme was 120,000 by native pore gradient PAGE. The enzyme was composed of four identical subunits with subunit molecular weights of 30,000 by SDS-PAGE. The Km values of the enzyme for L-threonine with and without AMP were 7.3 and 92 mM, respectively. There were 2 moles of pyridoxal phosphate and 16 moles of free -SH groups per 1 mole of enzyme. The enzyme was inhibited by $\alpha$-ketobutyrate, pyruvate, glyoxylate, and phosphoenol pyruvate(PEP) in the presence of AMP, yet stimulated by cAMP and ADP. For enzyme properties in comparison with S. marcescens, E. coli, and S. typhimurium enzyme, such as the PLP content, number of free sulfhydryl groups, and existence of ADP binding site, the S. marcescens enzyme was more similar to the S. typhimurium enzyme than the E. coli enzyme. Of the three enteric bacteria, the E. coli and S. typhimurium enzyme was increased the activity by ADP and cAMP, respectively, but only the S. marcescens enzyme was increased the activity by both ADP and cAMP. Therefore, the subtle differences in the properties between enzymes from the three enteric bacteria may represent minor structural differences among these enzymes and warrants further study.

• Journal of Life Science
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• v.25 no.5
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• pp.507-514
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• 2015

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250±20 μM and 1,300±50 μM, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.