• Journal of Sericultural and Entomological Science
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• v.41 no.3
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• pp.147-153
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• 1999

Chitinolytic enzymes such as ${\beta}$-N-acetylglucosaminidase are major hydrolases involved in insect molting. We have isolated, sequenced a CDNA encoding ${\beta}$-N-acetylglucosaminidase from the silkworm, Bombyx mandarina, and compared its sequence with genes encoding chitinolytic enyzmes from other sources. The insert DNA in the clone is 3,284 nucleotides long with an open reading frame of 1,788 nucleotides that encodes a protein of 596 amino acids with a molecular weight of 68.2 kDa. There is a 3’-untranslated region composed with 1.479 nucleotides and are several potential polyadenylation signals. The predicted amino acid sequence apparently contains a leader peptide of 23 amino acids. A search of the amino acids sequence databases for sequences similarities to other ${\beta}$-N-acetylglucosaminidases or ${\beta}$-N-acetylhexosaminidases. The highest similarity matched with the enzyme from B. mori, which has a sequence identity of 95%. On the other hand, the identity between the B. mandarina enzyme and those from M. sexta and human are 70% and 24%, respectively.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.109-113
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• 1998

In this study we tried to transform an antimicrobial peptide gene (Shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) into tobacco and potato plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified ie nucleotide sequence to increase antimicrobial activity. Transgenic tobacco plants were regenerated and their seeds were tested on the media containing kanamycin (500 mg/L). The results of PCR amplification and genomic Southern blot hybridization confirmed the integration of construct (tPAL5 promoter-Shiva-NOS-GUS-NOS) into chromosome. We observed that one of the transgenic tobacco plants showed chromosome rearrangement when integrated. In case of potato transformation, the efficiency of regeneration was maximized at the medium containing Zeatin 2mg/L, NAA 0.01mg/L, GA$_3$ 0.1mg/L. We also observed the high expression of GUS (${\beta}$-glucuronidase) enzyme which was located next to the terminator sequence of nopaline synthase gene (NOS) in the vascular tissue of stem, leaves of transgenic potatoes. This result suggested that a short sequence of Shiva gene (120 bp) and NOS terminator sequence might be served as a leader sequence of transcript when translated.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.63-70
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• 2017

A functional screen of 60,672 fosmid metagenomic clones amplified from marine sediment obtained from the Dokdo islets in Korea identified the gene EstES1, whose product, EstES1, displayed lipolytic properties on tributyrin-supplemented media. EstES1 is a 576 amino acid protein with a predicted molecular weight of 59.4 kDa including 37 N-terminal leader amino acids. EstES1 exhibited the highest sequence similarity (44%) to a carboxylesterase found in Haliangium ochraceum DSM14365. Phylogenetic analysis indicated that EstES1 belongs to a currently uncharacterized family of lipases. Within the conserved domain, EstES1 retains the catalytic triad that consists of the consensus penta-peptide motif, GESAG. EstES1 demonstrated a broad substrate specificity toward the long acyl group of ethyl esters (C2-C12), and its optimal activity was recorded toward p-Nitrophenyl butyrate (C4) at pH 9.0 and $40^{\circ}C$ (specific activity of 255.4 U/mg). The enzyme remained stable in the ranges of $60-65^{\circ}C$ and pH 9.0-10.5 and in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, EstES1 has potential for use in industrial applications involving high temperature, organic solvents, and/or alkaline conditions.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• BMB Reports
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• v.29 no.5
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• pp.429-435
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• 1996

To investigate the structure-antagonistic relationship of the cyclohexapeptide L-659,877, a selective NK-2 tachykinin receptor antagonist, seven analogues were chemically synthesized by a solid phase method. The agonistic and antagonistic activities of the analogues were evaluated by contraction assay using the smooth muscle of guinea pig trachea (GPT) containing the NK-2 receptor. It was shown that the aromatic ring of Phe at position 3 and the sulfur group of Met at position 6 in L-659,877 were essential for binding to the NK-2 receptor. Decrease in antagonistic activity of L-659,877 caused by substituting Leu for Nle at position 5 indicates that the ${\gamma}$ methyl group and side chain length of Leu plays an important role in its antagonistic action. Although the activity was slightly lower than L-659,877, cyclo $[{\beta}Ala^{8}]NKA(4-10)$ (analogue 1) showed potential antagonistic activity for the NK-2 receptor. It was confirmed that the expansion of the ring in L-659,877 by substitution of ${\beta}Ala$ for Gly at position 4 stabilized its conformation monitored by CD spectra. The results suggest that analogue 1 can be used as a new leader compound to design a more powerful, selective, and stable NK-2 receptor antagonist.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.33-33
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• 2001

본 연구는 한우 난포발달에 있어서 난포액 및 inhibin의 생리적 역할을 검토하기 위해 수행하였다. Anti-inhibin serum(AIS) 생산을 위해 사용된 항원은 porcine inhibin-$\alpha$-subunit 19~32의 peptide를 사용하여 adjuvant 용액을 1:3의 비율로 혼합하여 앙고라종 토끼 5두(체중 2.5kg)에게 주 2회 간격으로 8회 실시후 ELISA Leader로 항혈청의 역가를 확인하였다. 난포액(bFF; bovine follicular fluid)은 도축장에서 도축되는 한우 난소로부터 직경 1.0cm 이하의 난포로부터 회수하여 스테로이드를 제거하기 위해 10% chacoal solution(50 mg/$m\ell$, Norrit-A, Fisher Sci., USA)을 처리하여 45분간 배양후 원심분리후 상층액을 회수하여 실험에 공시하였다. 과립막세포의 체외배양을 위해 D-MEM용액(10% FCS와 antibiotics를 첨가)을 배양액으로 하여 1 $\times$ $10^{6}$ cells/$m\ell$로 조절하였다. 호르몬은 RIA 및 ELISA법으로 분석하였다. 그 결과 항원-항체반응은 항원처리후 24일째부터 항체가를 확인할 수 있었으며 52일째에서 높은 항원-항체반응을 보였다. 한편, 난포크기별 난포액의 progesterone 및 Estradiol-l7$\beta$을 농도를 분석한 결과, Progesterone 난포크기가 직경 1.0 cm부터 유의적으로 증가하기 시작하여 직경 2.0cm의 난포액에서는 높은 progesterone이 존재하고 있는 것으로 나타났다. 이에 반해, 난포크기별 난포액중 estradiol 17$\beta$농도는 직경 1.0cm구에서 가장 높게 나타났다. 난포직경이 1cm일 경우에 난포액내 etradiol-17$\beta$가 가장 많이 존재하고 있음이 나타났다. 과립막세포의 체외배양에서 배양 24시간에서는 과립막세포의 progesterone분비는 약 40ng/1$\times$$10^{6}$ cell/well/$m\ell$ 전후로 나타났으며 bFF 5%, AI 5% 및 bFF＋AI 첨가구에 따라 유의적인 차이는 없었다 반면에 48시간배양구에서는 24시간에 비해 유의적으로 높게 분비되었으며, bFF 5%처리구와 bFF5%＋AI5%처리구에서는 progesterone을 대조구보다 유의적으로 억제되었으나 AI 5%단독처리구에서는 대조구와 큰 차이가 없었다. 한편, 각 세포질을 SDS-PAGE로 분리하여 nitro cellulose membrane에 transfer하여 Western blotting법에 의해 검토한 결과, 직경 1.0 cm의 성숙난포의 granulosa cell에 특이하게 Inhibin이 존재하고 있음이 확인되었다. 이상의 결과로, 한우에 있어서 성숙난포에 존재하는 Inhibin은 난포발달 및 난포세포의 스테로이드호르몬합성에 중요하게 관여하고 있는 것으로 사료된다.