• The Korean Journal of Mycology
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• v.26 no.3 s.86
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• pp.354-360
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• 1998

We isolated three amplified DNA fragments from P. sajor-caju by Polmerase chain reaction (PCR) using the chithin synthase specific primers. Since the sequence analysis of the these fragments showed significant homology to the other known chitin synthase gene, we regarded these cloned fragments as PsCHS1, PsCHS2, and PsCHS3 according to their size. The PsCHS3, which showed the highest sequence homology (83% identity in amino acid level with ChsI of Rhizopus oligosporus in conserved region), was selected to see expression pattern of the corresponding gene. The result of RT-PCR using internal primer of the PsCHS3 fragment revealed that PsCHS3 gene was only expressed in cap and mycelium but not in stipe. In order to see whether the PsCHS3 gene was to be induced by wounding, the comparison of the mRNA level of this gene between wounded and unwounded mature cap showed at least two times induction of this gene by wounding treatment.

• Journal of Life Science
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• v.12 no.3
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• pp.264-273
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• 2002

We have isolated and artificially expressed three cDNA clones of Capsicum annuum PR5 genes for elucidating the antifungal activity against Phytophthora capsici which contracted a hot pepper root rot in field condition. Three divergent PR5 proteins from hot pepper were designated as CAPR5-1 and CAPR5-2 from susceptible cultivar (Subicho) as well as CAPR5-3 from resistant cultivar (CM331) in response to P. capsici. The cDNA similarity was found over 80% of identity among the three CAPR5s, and deduced amino acid sequence was characterized that all of CAPR5s contained 16 cysteine residues which possibly had a significant role in the structural formation. The result of genomic DNA blot showed that CAPR5-1 and CAPR5-2 existed as single copy in the Subicho genome. Three recombinant CPARs in E. coli were identified by SDS-PACE, and each expressed protein was treated on the PDA medium which contained cultured pathogens. Although three CAPR5 proteins did not affected the hyphal growth of Glomerella glycines and Colletotrichum fagenarium, CAPR5-1, CAPR5-2, and CAPR5-3 showed a specific antifungal activities against P. capsici.

• Journal of Life Science
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• v.19 no.2
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• pp.284-288
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• 2009

The innate immune system is important for the first line of host defence against infectious agents, which have penetrated the mechanical barriers. Mannose-binding lectin (MBL or mannan-binding protein, MBP) is a serum protein that is synthesized in the liver as a part of the acute phase response. MBL binds to carbohydrate structures presented by a wide range of pathogenic bacteria, viruses, fungi, and parasites. MBL is synthesized as a monomer that has a carboxy-terminal carbohydrate recognition domain, a neck region and a collagen region. Low MBL level was reported to be the most frequent immuno-deficiency syndrome. Although extensive studies have yielded detailed information on the structure of MBL, functions of the MBL complex are not fully understood yet. We, here, present cloning process of MBL cDNA from the rat liver and production of truncated recombinant MBL protein using a bacterial expression system in order to produce anti-MBL polyclonal antibody. Anti-MBL polyclonal antibody was raised in a New Zealand rabbit and its affinity was tested against recombinant protein using western blot technique. MBL cDNA, recombinant protein and anti-MBL antibody could be used as great arsenals to dissect cellular biochemistry of MBL.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.203-208
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• 2000

A Baczllus circdans KCTC3004 $\alpha$-amylase gene contained in a recombinant plasmid pAL850 was transferred into a new shuttle vector plasmid pALSIlI by ligating linearlzed DNAs of pUC19 and pUB110. B. subtilis RM125 and B. megatenurn ATCC14945 transfonned with pALS111 produced the $\alpha$-amylase substantially Most of the enzyme was produced during the exponential growth period. The maxiinurn activities of the $\alpha$-amylase produced by the Bucillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125(pALS111) enzyme showed the actlvicy 95 times higher than that of the gene donor cells, followed by the B, nzegaterium ATCC14945(pALSlll) enzyme with activity 34 limes higher than that of the gene donor cells. While E coli secreted about 10% of the produced enzyme, B. subtilis excreted the enzyme inlo the medium wholly and B. megaterirun about 98% ofthe total product. The plasmid pALSI11 was quite stable inB. nzegaterium (92%), inoderately stable in B. subtilis (76%), but was unstable in E. coli (38%). The SDS-PAGE and zymogram of this enzyme produced in E. coli(pALS111), B. subtilis( pALS111) or B. megateril~m (pALS111) indicated a molecular weight of 55,000. The enzymes overproduced in three different host cells hydrolyzed starch to produce mainly maltoaiose and mallooligosaccharides.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.142-148
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• 2006

In order to develop chitosanase for the production of chitosan oligosaccharides, a chitosanase-producing bacterium was isolated from the traditional fermented soybean, Meju, and identified as Bacillus amyloliquefaciene MJ-1. The cloned chitosanase gene, 825 bp in size, encoded a single peptide of 274 amino acids with a estimated molecular mass of 30.9 kDa. The deduced amino acid sequence showed significant homology with microbial chitosanases. The recombinant chitosanase was expressed in Escherichia coli upon induction with isopropyl-D-thiogalactopyranoside, and purified using $Ni^{2+}-NTA$ agarose column chromatography. The maximal activity of the recombinant chitosanase is at pH 5.0 and $60^{\circ}C$. The recombinant chitosanase is stable between pH 5.0 and pH 7.0 at $37^{\circ}C$ for 30 min, and more than 75% of the activity still remain at $80^{\circ}C$ for 30 min incubation.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.255-264
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• 2018

This experiment was conducted to analyse the effects of flavone supplementation on the preimplantation development of in-vitro produced porcine embryos. During in-vitro development, immature oocytes and early embryos were exposed to different concentrations of flavone (0, $1{\mu}M$, $25{\mu}M$, $50{\mu}M$, and $100{\mu}M$ respectively). Results showed that $100{\mu}M$ of flavone significantly reduced the intracellular ROS levels of oocytes accompanied with a significant rise in GSH level. In parthenogenesis, no significant change was observed in the cleavage rates whether flavone was supplemented in IVM or IVC media. In IVM supplemented group, the blastocyst development rate was significantly enhanced by $1{\mu}M$ concentration than other groups (51.5% vs. 41.3%, 44.0%, 36.3%, 31.7%; P<0.05) respectively. However, in IVC group $1{\mu}M$ concentration significantly improved the blastocysts production than $50{\mu}M$ and control groups (50.0% vs. 40.5%, 38.0%; P<0.05) respectively. Following nuclear transfer, the cleavage rate of IVM group was significantly more in $1{\mu}M$ than $50{\mu}M$ and $100{\mu}M$ groups (92.9% vs. 89.7%, 87.8%; P<0.05), followed by similar pattern of cloned blastocysts production being significantly higher in $1{\mu}M$ group than $50{\mu}M$, $100{\mu}M$ and control groups (16.8% vs. 9.0%, 7.1%, 12.8%; P<0.05) respectively. In IVC group, $1{\mu}M$ concentration resulted in significantly higher cleavage rate than $25{\mu}M$ and $50{\mu}M$ groups (91.7% vs. 87.8%, 88.8%; P<0.05) respectively. However, the blastocysts production was significantly higher in $100{\mu}M$ group than others (26.2% vs. 13.6%, 14.0%, 18.2%; P<0.05) respectively. The optimal concentrations of flavone significantly enhanced the percentages of ICM:TE than control group (43.8% vs. 37.6%; P<0.05) accompanied with significantly higher expression levels of reprogramming related genes. In conclusion, the optimal concentrations of $1{\mu}M$ during IVM and $100{\mu}M$ during IVC can significantly improve the production of porcine in-vitro embryos.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.45-51
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• 2014

A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.

• Applied Biological Chemistry
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• v.42 no.4
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• pp.293-297
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• 1999

The pts operon, which encodes several factors in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Escherichia coli, has multiple promoters which respond to different signals to facilitate quick adaptation to changes in growth conditions. The influence of an 1 kbp DNA region upstream of the pts P0 promoter on pts expression was studied in vitro by employing the DNA templates containing both P0 and P1 promoter with or without the 1 kbp upstream DNA region for in vitro transcription assay. The 1 kbp DNA region upstream of the pts P0 promoter, however, had no effect on pts transcription in vitro. The intracellular concentration of cAMP was measured when cells were grown in the presence of glucose, mannose, or mannitol. The transcription of P0 was increased maximally in the presence of glucose even though the concentration of cAMP in the condition was lowest while the transcription from the P1b was highest when cells were grown in the presence of mannose or mannitol even though the intracellular concentration of cAMP was lower than cells grown in the absence of the sugar. These results suggest the possibility of the existence of a glucose inducible repressor specific for the P0 promoter and a second repressor that is inducible by glucose, mannose and mannitol specific for the P1 promoter.

• Journal of Life Science
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• v.14 no.5
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• pp.834-839
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• 2004

To improve and oil degrading activity, the lipase gene from Pseudomonase sp. was transformed into Klebsiella sp. KCL-l, an oil degrading bacterium. The selected trasformant was named as a KCL-1/pET-Lip. The surface tension of culture broth of KCL-1/pET-Lip was decreased to 33 dyne/cm from 55 dyne/cm using 4% (v/v) soybean oil as sole carbon source. The surface tension were 44 and 37.5 dyne/cm, to 2% (w/v) glucose and 4% (v/v) kerosene medium, respectively. The emulsification activity of the biosurfactant solution containing lipase of KCL-l/pET-Lip improved better than wild type KCL-l. The soybean oil was most efficient carbon source and substrate for surface activity and emulsification activity of KCL-1/pET-Lip. The expression of lipase was confirmed by SDS-PAGE.