• 한국축산식품학회지
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• 제21권4호
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• pp.367-373
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• 2001

MFGM 당단백질의 하나인 PAS-7을 GPC 및 affinity chromatography에 의해 정제하여 2-AB로 형광 표식한 후, anion-exchange column 및 reversed-phase column을 이용해 5개의 성분을 분리하였다. 그 중 가장 상대량이 많은 성분 e에 대하여 RAAM2000을 이용한 당쇄구조 해석을 실시하여, 성분 e는 RAAM2000 GPC에 의하여 4개의 성분으로 분리되어 각각 calibration standard 12.10, 8.88, 5.84 및 4.86GU의 용출위치에 검출되었다. 이 용출위치와 당쇄구조는 livrary의 component-7457과 일치하며, 12.2GU의 크기로 분자량은 1788로 판단되며 library의 당쇄와 약 85%의 확률로 일치했다. 그 결과 성분 e의 당쇄구조는 환원말단에 $\alpha$1-6결합된 fucose를 1개 함유하며, core부분의 비환원말단에 N-acetyllactosamine branch를 2개 함유한 전형적인 biantennary 당쇄구조인 것으로 추축되어, 이전 HPLC, acetolysis, sequential exoglycosidase 소화, NMR분석에 의해 보고된 성분 7N1A의 구조와 일치함으로써, OGS RAAM2000을 이용한 PAS-7의 당쇄구조 해석의 유용성이 증명되었다.

• Journal of Dairy Science and Biotechnology
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• 제21권2호
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• pp.138-147
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• 2003

We here analyzed and proposed the structures of the N-linked sugar chains of PAS-7 from bovine milk fat globule membrane. The N-linked sugar chains were liberated from PAS-7 by hydrazinolysis and, after modifying the reducing ends with 2-aminopyridine (PA), were separated into one neutral (7N,55%) and two acidic (7M mono-, 43%; 7D, di-, 2%) sugar chain roups. The latter were converted into neutral groups (7MN and 7DN) by sialidase digestion. The structure of each of these PA-neutral sugar chains was determined by sugar analysis, sequential exoglycosidase digestion, partial acetolysis, and 1H-NMR spectroscopy. The results show that the 10 sugar chains were of the biantennary complex type with and without fucose. The structure of 7N2A one of the major sugar chains, was proposed as; [structure: see text] A structural comparison between PAS-6 and -7 indicated that although they shared the same protein core, their sugar moiety was markedly different, involving the existence of a different pathway during the post-transcriptional modification.

• 한국식품과학회지
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• 제29권4호
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• pp.630-634
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• 1997

N-glycanase처리에 의해 얻어진 1-amino-oligosaccharide를 FMOC-Cl로 표식 하는 방법을 확립하였다. 이 방법에 의해 얻어진 FMOC-표식 oligosaccharides는 기존의 방법보다 약 4배의 감도를 나타내었다. Amido 80 column을 사용한 분석에 의하여, Man 5-9 GlcNAc 2 amines의 5개의 성분은 각각 분리되어 용출 되었으며, 회수율은 기존의 방법과 거의 같았다. 1-amino-oligosaccharides는 0.05 pmol에서 1.5 pmol 사이에서 직선적인 관계를 나타내었으며, 2-aminopyridine에 의한 표식과 비교하였을 때, 안정된 화합물을 형성하고 있으며, 감도가 높아 미량의 oligosaccharides의 분석에 매우 적합한 것이 확인되었다.

• 한국식품저장유통학회지
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• 제15권5호
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• pp.617-621
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• 2008

Sequential passes through $Sephadex^{TM}$ columns were used to select phages that displays ligands for dextran ($\alpha$-1,6 linked linear chains) from a phage antibody library. Those phages that bound to the $Sephadex^{TM}$ in each iteration were replicated in E. coli. A phage preparation isolated on the third round selection produced 5.4 nephelos turbidity units (NTU) in a dextran specific immunonephelometric assay, a 2.2 fold higher value than the phage preparation from the first round selection. This phage gave $72\;{\pm}\;10$ normalized intensity (N.I.) in a dip-stick assay against high molecular size dextran (T2000, $2\;{\times}\;10^6) and significantly lower color ($30\;{\pm}\;6$ N.I.) against low molecular size dextran (T10, $10^4$). The presence of an Fab insert in each of these phages was confirmed using a $\beta-galactosidase linked assay and polymerase chain reaction.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권2호
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• pp.136-140
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• 2000

A sensitive and quantiative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glconproproteins. Inthis study, n-linked oligosaccharides of human IgG and bovine transferin were analyzed for the evaluation of the methydrate moiety ofthe method. Chrbohydrate moiety of glycoprotein was relased by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent bye, 2-aminobenzamide, for the enhancement of detection sensitivity. sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column(MonoQ) followed by the treatment with sialidase. Enzymatically desiayated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligosaccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.383-391
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• 2008

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

• 한국식품영양과학회지
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• 제31권2호
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• pp.177-183
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• 2002

대두 및 대두발효식품인 청국장과 된장의 cerebroside 구성성분인 지방산, 당 및 장쇄 sphingoid base들을 GC-MS로 분석하였다. Cerebroside 산-가수분해물의 지방산들을 O-TMS methylester 유도체로 만들어 분석하였으며 대두 및 청국장의 경우 2-hydroxyhexadecanoic acid(16 : 0h)가 각각 52.2% 및 62.0%로 가장 많았고 2-hydroxydocosanoic acid(22 : 0h) 및 2-hydroxytetracosanoic acid(24 : 0h)도 15.0~17.6%이었다. 그러나 된장의 경우 16 : 0h는 10.2%로 많은 감소를 보인 반면 24 : 0h는 40.9%, 22 : 0h는 23.4% 었으며 23 : 0h, 25 : 0h 및 26 : 0h도 대두 및 청국장에 비하여 많았다. Cerebroside 산-가수분해물의 장쇄 sphingoid base들은 N-acetyl-O-TMS 유도체로 분석하였으며 대두 및 청국장에서 4- trans, 8-trans-sphingadienine (d18 : 2 $\Delta$$^{4trans, 8trans}$) 는 59.9% 및 44.5%이었으며 4-hydroxy-8-trans, cis-sphingening (t18 : 1$\Delta$$^{8trans or cis}$)는 20.9% 및 35.9% 이었고 된장 cerebroside 역시 dl8 : 2$\Delta$$^{4trans, 8trans}$가 가장 많았다. 그러나 phytosphingosine(t18 : 0) 및 sphingosine 이 성질체(d18 : 1)들은 거의 보이지 않았다. 또한 된장의 경우 오랜 기간 미생물들에 의하여 발효되었지만 Aspergillus 속들의 cerebroside가 가지는 것으로 알려진 2-hydroxyoxtadec-3-enoic acid(18 : 1h) 및 9-methyl-4,8-sphingadienine 등은 발견되지 않았다. 대두, 청국장 및 된장 cerebroside의 당유도체 mass spectrum 분석 결과 99%이상이 glucose 1번 탄소가 ceramide backbone에 결합된 monoglucocerebroside 인 것으로 나타났다.