• 한국미생물·생명공학회지
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• 제26권1호
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• pp.68-75
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• 1998

A. niger의 GOD(Glucose Oxidase) 대량생산과 효율적인 분비를 protein의 대량생산에 많이 사용되는 strain인 S. cerevisiae에서 시도하였다. S. cerevisiae의 ADH1과 GAL 10 promotor, 그리고 ${alpha}$-MF signal sequence와 A. oryzae의 ${alpha}$-amylase signal sequence 및 S. cerevisiae의 GAL7과 A. niger의 GOD terminator를 이용하여 4개의 expression vector를 합성한 후 S. cerevisiae 2805에 auxotroph 방법으로 형질변환시켰다. 변이체들을 배양하여 세포내와 세포외의 GOD활성도를 분석한 결과 GAL 10 promotor가 삽입된 pGAL변이체들이 ADH1 promotor가 삽입된 pADH 변이체들 보다 GOD 생산성이 높았다. GAL 10 promotor와 A. oryzae의 ${alpha}$-amylase signal sequence가 삽입된 pGALGO2에서 115시간 배양시 GOD의 생산이 가장 높았다($GOD_{total}$: 10.3 unit/mL, $GOD_{ex}$: 8.7 unit/mL). 이 수치는 같은 promotor인 GAL 10 promotor와 ${alpha}$-MF signal sequence가 삽입된 pGALGO1보다 3배정도 높다. 이 결과는 ADH 1 promotor를 사용하였을 경우에도 일치하였다. 또한 A. oryzae의 ${alpha}$-amylase signal sequence가 S. cerevisiae의 ${alpha}$-MF signal sequence보다 GOD를 더 효과적으로 분비시켰다. 상기 결과로 미루어 보면 signal sequence가 단백질의 분비 외에도 단백질 합성에도 많은 영향을 주는 것으로 추측된다. pGALGO1과 pGALGO2의 GOD분비효율은 각각 89%, 84%이었다. S. cerevisiae에서는 일반적으로 과당화가 일어나기 때문에 S. cerevisiae에서 합성된 재조합 GOD의 분자량은 250 kDa으로 A. niger의 GOD(170 kDa)보다 더 컸다.

• KSBB Journal
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• 제14권4호
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• pp.476-481
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• 1999

본 연구에서 갈락토즈를 거의 사용하지 않고 glucose repression 정도가 줄어든 변이주를 이용하여 발현최적화를 수행하였다. 두 균주에서의 GAL promoter에 의한 외래단백질 생산시 glucose repression 정도에 대해 조사하였는데 대조해 Y2805는 glucose가 다 소비된 후 2~3시간 지난 후 발현이 시작되나 변이주 Y334는 약 0.5g/L 글루코즈 농도에서 25%정도의 발현이 이루어짐에 따라 변이주 Y334는 GAL promoter에 미치는 glucose repression정도가 매우 약한 장점을 확인하였다. GAL promoter에 의한 외래 단백질 생산시 발현을 위한 최적 갈락토즈 농도를 조사하였는데, Y3805는 3% 까지의 높은 갈락토즈 농도에서, 변이주 Y334는 1%정도의 낮은 갈락토즈 농도에서 각각 최대 발현량을 보였으며 변이주 Y334는 특히 0.01%정도의 낮은 갈락토즈 농도에서도 최대 발현의 60%량을 발현하였고 오히려 높은 갈락토즈 농도에서는 성장장애 현상을 보였다. 두 균주를 이용하여 배지중 pH가 외래 단백질 생산에 미치는 영향을 조사하였는데 두 균주 모두 pH 5근처에서 최대 발현량을 보임을 알 수 있었다. GAL promoter에 의한 외래 단백질 생산시 글루코즈와 갈락토즈, 에탄올의 소비속도를 조사하였는데, 글루코즈와 에탄올의 소비속도는 거의 비슷하였으나 갈락토즈 소비속도는 Y2805는 0.1232 g/L/hr/O.D.이고, 변이주 Y334는 0.0131g/L/hr/O.D. 이다. 또한 두균주의 분비효율을 조사하였는데 Y2805는 발효후반부에 총 생산 albumin중 약 70%는 분비되었고 30%는 cell 내 위치하는 것을 알 수 있었고 Y334의 경우에는 발효후반부에 총 생산 albumin중 50%가 cell 밖으로 분비되고 50%는 cell 내에 존재하는 것으로 확인되었다. 이는 Y334의 해결해야 할 단점으로 생각 되어지며 이러한 문제의 해결을 위해 albumin 생산성이 2~3배 증가된 초분비 S. cerevisiae 돌연변이주 개발이 현재 진행중이다.

• 대한수의학회지
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• 제54권4호
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• pp.209-218
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• 2014

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), reflects pathophysiologic steps in MS such as the influence of T cells and antibodies reactive to the myelin sheath, and the cytotoxic effect of cytokines. Galectin-9 (Gal-9) is a member of animal lectins that plays an essential role in various biological functions. The expression of Gal-9 is significantly enhanced in MS lesions; however, its role in autoimmune disease has not been fully elucidated. To identify the role of Gal-9 in EAE, we measured changes in mRNA and protein expression of Gal-9 as EAE progressed. Expression increased with disease progression, with a sharp rise occurring at its peak. Gal-9 immunoreactivity was mainly expressed in astrocytes and microglia of the central nervous system (CNS) and macrophages of spleen. Flow cytometric analysis revealed that $Gal-9^+CD11b^+$ cells were dramatically increased in the spleen at the peak of disease. Increased expression of tumor necrosis factor (TNF)-R1 and p-Jun N-terminal kinase (JNK) was observed in the CNS of EAE mice, suggesting that TNF-R1 and p-JNK might be key regulators contributing to the expression of Gal-9 during EAE. These results suggest that identification of the relationship between Gal-9 and EAE progression is critical for better understanding Gal-9 biology in autoimmune disease.

• Asian-Australasian Journal of Animal Sciences
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• 제33권11호
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• pp.1837-1847
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• 2020

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media - advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

• 한국수정란이식학회지
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• 제29권4호
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• pp.333-337
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• 2014

To compensate for the critical shortage of human organs for allotransplantation, xenotransplantation studies using genetically modified pigs are being performed in Korea. Two types of pigs that are used are ${\alpha}1,3$-galactosyltransferase gene knockout (GalT KO) pigs and GalT KO+hCD46 (human complement regulatory protein) pigs. The present study measured the gestation time, birth weight, daily growth rate, and heart weight of both kinds of transgenic minipigs. The gestation period for both types of pigs was 117~119 days. There was no difference in the body weight of GalT KO (-/+) and GalT KO (-/-) piglets, but GalT KO+hCD46 ($-^{hCD46+}/+$) pigs were significantly heavier at birth than were GalT KO+hCD46 ($-^{hCD46+}/-^{hCD46+}$) pigs. During the first 10 weeks of life, the daily weight gain of GalT KO+hCD46 ($-^{hCD46+}/-^{CD46+}$) piglets, which are considered the optimal type for xenotransplantation, was 0.19 kg. The weight of hearts from GalT KO piglets up to two months of age was affected more by body weight than by age. Transgenic pigs showed no differences in gestation period or reproductive ability compared with normal pigs. These results comprise basic data that may be used in xenotransplantation studies and transgenic animal production in Korea.

• Journal of Ginseng Research
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• 제48권2호
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• pp.202-210
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• 2024

Background: Panax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. Methods: P. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. Results: Our ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar K_D values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. Conclusion: P. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

• Bulletin of the Korean Chemical Society
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• 제24권3호
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• pp.339-344
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• 2003

The conformation of synthetic oligosaccharide can be elucidated by employing molecular modeling and highfield proton NMR (nuclear magnetic resonance) spectroscopy. Information with respect to the composition and configuration of saccharide residues and the sequence and linkage positions of the oligosaccharide can be obtained by employing a variety of one- and two-dimensional NMR techniques and molecular modeling. These techniques are also useful in establishing the solution conformation of the oligosaccharide moiety. This study is focused on the elucidation of linkage positions of synthetic trisaccharides, Gal(β1-4)Glc(β1-3)Glc, Gal(β1-4)Glc(β1-4)Glc and Gal(β1-4)Glc(β1-6)Glc.

• 한국미생물·생명공학회지
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• 제40권4호
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• pp.348-355
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• 2012

본 연구에서는 S. cerevisiae와 P. pastoris에서 bovine pancreatic (bp-) DNase I의 과발현과 재조합 DNase I의 특성을 조사하였다. bp-DNase I 유전자는 GAL10 promoter, $MF{\alpha}$, GAL7 terminator 사이에 삽입하여 재조합 plasmid인 pGAL-$MF{\alpha}$-DNaseI (6.4 kb)를 구축하였다. 그리고 bp-DNase I 유전자를 AOX1 promoter, $MF{\alpha}$, AOX1 terminator 에 삽입하여 재조합 plasmid인 pPEXI (8.8 kb)를 구축하였다. 재조합 plasmid인 pGAL-$MF{\alpha}$-DNaseI과 pPEXI를 각각 S. cerevisiae와 P. pastoris 숙주세포에 형질전환시켰다. 형질전환된 효모세포들을 galactose와 methanol 배지에서 $30^{\circ}C$, 48시간 배양하면 bp-DNase I은 대부분이 배양 상등액으로 과발현되었다. P. pastoris 형질전환체는 배양 상등액에서 45.5 unit/mL의 DNase I 활성을 보였으며, 반면에 S. cerevisiae 형질전환체는 37.7 unit/mL의 DNase I 활성을 보였다. 또한 DNA 분해 특성을 조사한 결과, P. pastoris 재조합 DNase I으로 기질 DNA(calf thymus)를 처리하였을 때 1분 이내 DNA가 분해되는 것을 확인할 수 있었으며 이는 상업용 bp-DNase I과 S. cerevisiae 재조합 DNase I으로 처리했을 때보다 빠른 분해 패턴을 보였다.

• Bulletin of the Korean Chemical Society
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• 제29권9호
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• pp.1755-1760
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• 2008

Energy minimization and conformational studies of molecular ions generated by ESI (electrospray ionization) tandem mass spectrometry (MS/MS) can be used for the discrimination of stereoisomeric permethylated and sodium cationized trisaccharides. Sets of fucose-containing trisaccharides having different internal and terminal linkages have been synthesized to analyze the reducing terminal linkage positions using BT and IT fusion approaches. A detailed investigation has been undertaken on the conformational behaviors of four trisaccharide fragments from human milk and blood group determinants of Type 1 and Type 2, namely Fuc($\alpha$1- 3)Gal($\beta$1-3)GalNAc and Fuc($\alpha$1-3)Gal($\beta$1-X)GlcNAc where X = 3, 4 and 6 using molecular modeling methods. Three dimensional rigid and adiabatic phi-psi-energy maps (Surfer program) describing the energy as a function of rotation around corresponding glycosidic linkages were calculated by SYBYL molecular modeling and MM4 force field programs conjunction with cleavage energies of ESI MS/MS for the side group orientations. This approach predicted conformational behaviors exhibited by isomer saccharides for future applications on biologically active glycoconjugates and to exploit a faster method of synthesizing a series of structural isomeric oligosaccharides.

• 상하수도학회지
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• 제20권6호
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.