Name: 인간의 장내 미생물총(microbiota)에서 C-배당체 대사 효소를 특성화하기 위한 구조 생물학 및 분석 화학 접근법 (동영상) | JoVE Journal
Uploaded: 2025-05-23T12:50:00.000+00:00
Duration: 13 min 35 s
Description: 학습하세요 인간 장내 미생물총의 C-배당체 대사 효소를 구조 생물학 및 분석 화학 기법으로 특성화하는 방법을. 단백질 정제, 결정학, LC-MS/MS 및 NMR을 활용하여 효소 활성과 구조를 종합적으로 분석합니다.

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5 minutes

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<li>Karpinska, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+degradation+of+MLK3+by+a+novel+CEP1347-VHL-02+PROTAC+compound+limits+the+oncogenic+potential+of+TNBC%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">Selective degradation of MLK3 by a novel CEP1347-VHL-02 PROTAC compound limits the oncogenic potential of TNBC.</a> J Med Chem. 67 (17), 15012-15028 (2024).</li>
</ol>

<table><tbody><tr><td>2,6-디클로로인도페놀 (DCPIP)</td><td>Solarbio Science and Technology</td><td>D6350</td><td></td></tr><tr><td>2-메르캅토에탄올(&beta;-ME)</td><td>시그마-알드리치</td><td>08168</td><td></td></tr><tr><td>&nbsp; 5 &times; 단백질 로딩 염료</td><td>Sangon Biotech</td><td>C508320</td><td></td></tr><tr><td>아세트산</td><td>Shanghai MACKLIN</td><td>A111247</td><td></td></tr><tr><td>Acetonitrile</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>A12147</td><td></td></tr><tr><td>Bruker Avance III HD 700 MHz NMR 분광계</td><td>Bruker</td><td>AVANCE III HD 700MHz</td><td></td></tr><tr><td>Bruker Ultrashield 400 Plus 400MHz</td><td>Bruker</td><td>AVANCE III 400 MHz</td><td></td></tr><tr><td>원심분리기 5424R</td><td>Eppendorf</td><td>022620601</td><td></td></tr><tr><td>Chirascan; 플러스 CD 분광계</td><td>응용 광물리학</td><td>Chirascan&trade; &nbsp;</td><td></td></tr><tr><td>Daidzein</td><td>Shanghai Yuanye Bio-Technology</td><td>B20227</td><td></td></tr><tr><td>Daidzin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20226</td><td></td></tr><tr><td>Dithiothreitol(DTT)</td><td>Solarbio Science and Technology</td><td>D8220</td><td></td></tr><tr><td>E. coli BL21 (DE3) 유능 세포</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>EC0114</td><td></td></tr><tr><td>Genistein</td><td>Shanghai Yuanye Bio-Technology</td><td>S31565</td><td></td></tr><tr><td>Genistein-8-C-glucoside</td><td>Shanghai Yuanye Bio-Technology</td><td>BP2095</td><td></td></tr><tr><td>Genistin</td><td>Shanghai Yuanye Bio-Technology</td><td>S31591</td><td></td></tr><tr><td>포도</td>당 <td>시그마-Aldrich</td><td>G8270</td><td></td></tr><tr><td>GraphPad 프리즘 9</td><td>GraphPad 소프트웨어</td><td>버전? Prism 9</td><td>Km 및 kcat 운동 매개변수 계산</td></tr><tr><td>HiLoad Superdex 200pg 분취용 SEC 컬럼</td><td>Cytiva</td><td>28989335</td><td></td></tr><tr><td>HisTrap HP His 태그 단백질 정제 컬럼</td><td>Cytiva</td><td>17524802</td><td></td></tr><tr><td>Q FF 음이온 교환 크로마토그래피 컬럼</td><td>Cytiva</td><td>17515601</td><td></td></tr><tr><td>HKL-3000</td><td>HKL research Inc</td><td>&nbsp; 버전 723</td><td></td></tr><tr><td>HPLC 컬럼 TC-C18(2), 170&Aring;, 5 &마이크로; m, 4.6 x 250 mm</td><td>Agilent Technologies</td><td>518925-902</td><td></td></tr><tr><td>이소 프로필 &beta; -D-1-thiogalactopyranoside? IPTG)</td><td>시그마-알드리치</td><td>PHG0010</td><td></td></tr><tr><td>유당</td><td>시그마-알드리치</td><td>17814</td><td></td></tr><tr><td>LC-20AT, 고성능 액체 크로마토그래피</td><td>Shimadzu</td><td>LC20AT</td><td></td></tr><tr><td>맥아당</td><td>시그마-알드리치</td><td>47288</td><td></td></tr><tr><td>말토트리오스</td><td>시그마-알드리치</td><td>443713</td><td></td></tr><tr><td>염화망간</td><td>Shanghai Yuanye Bio-Technology</td><td>S31112</td><td></td></tr><tr><td>메탄올</td><td>써모 피셔 과학&무역;? Invitrogen</td><td>M23142</td><td></td></tr><tr><td>NAD+</td><td>Solarbio 과학 기술</td><td>N8110</td><td></td></tr><tr><td>Olympus SZX12 실체 현미경</td><td>OLYMPUS&nbsp;</td><td>SZX7</td><td></td></tr><tr><td>페닐메틸설포닐 플루오라이드(PMSF)</td><td>시그마-알드리치</td><td>P7626&nbsp;</td><td></td></tr><tr><td>Phoenix</td><td>Phoenix 개발팀</td><td>1.21.2</td><td>Phenix는 단백질 결정 구조 측정을 위한 포괄적인 소프트웨어 제품군으로, SAD(Single-wavelength Anomalous Dispersion) 및 MR(Molecular Replacement) 방법을 Phaser 소프트웨어와 통합합니다.</td></tr><tr><td>준비 고성능 액체 크로마토그래프</td><td>Waters</td><td>2545/2998</td><td></td></tr><tr><td>단백질 결정 스크리닝 키트</td><td>Hampton Research</td><td>HR2-110/HR2-112/HR2-144/HR2-126/HR2-098</td><td></td></tr><tr><td>Puerarin</td><td>Shanghai Yuanye Bio-Technology</td><td>B20446</td><td></td></tr><tr><td>쉐이킹 인큐베이터</td><td>Shanghai Zhichu Instrument Co., Ltd</td><td>&nbsp; ZQZY-AF8</td><td></td></tr><tr><td>뱀 가죽&무역; 투석막, 10 kDa MWCO, 35 mm</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>88245</td><td></td></tr><tr><td>가용성 전분</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Sorvall LYNX 6000 초고속 원심분리기</td><td>Thermo Fisher Scientific&trade;</td><td></td><td></td>75006590</tr><tr><td>SpectraMax i3x 멀티모드 마이크로플레이트 리더</td><td>분자 장치</td><td>SpectraMax i3x&nbsp;</td><td></td></tr><tr><td>SpectraMax QuickDrop 마이크로 볼륨 분광 광도계 사양</td><td>분자 장치</td><td>QuickDrop</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S5016</td><td></td></tr><tr><td>SunFire C18 OBD Prep Column</td><td>Waters</td><td>186002741</td><td></td></tr><tr><td>Thermo Fisher Scientific Q EXACTIVE</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>유비퀴틴 유사 프로테아제 1</td><td>Thermo Fisher Scientific&trade;? Invitrogen</td><td>12588018</td><td></td></tr><tr><td>초저온 냉동고</td><td>Thermo Fisher Scientific</td><td>905GP-ULTS</td><td></td></tr><tr><td>진공 원심 농축기</td><td>베이징 JM Technology</td><td>CV600</td><td></td></tr><tr><td>Vitexin</td><td>Sigma-Aldrich</td><td>49513</td><td></td></tr></tbody></table>

인간의 장내 미생물총(microbiota)에서 c-배당체 대사 효소를 특성화하기 위한 구조 생물학 및 분석 화학 접근법

Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota

에 설명된 절차를 따릅니다. 그림 1, 먼저 DgpA, DgpB 및 DgpC 유전자를 E. coli BL21(DE3) 세포. DgpB와 DgpC는 안정한 효소 복합체(DgpB/C 복합체)를 형성하기 때문에10,26,27,34, 이 두 단백질을 발현하는 세포를 공동 정제를 위해 결합했습니다. 세포 용해 후, 상층액을 48,000 x에서 원심분리에 의해 수집하였다g 4 °C에서 40 분 동안 니켈 친화성 크로마토 그래피를 적용했습니다. 결합된 단백질은 완충액 A(그림 2A, 2<s...

인간의 장내 미생물총(microbiota)에서 C-배당체 대사 효소를 특성화하기 위한 구조 생물학 및 분석 화학 접근법

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While ...

structural-biology-analytical-chemistry-approaches-for-characterizing

Research

연구

JoVE Journal

Biochemistry

생화학

면역 기관과 세포의 glycosphingolipid 항원의 표현 프로필에 대한 통찰력을 얻을 수있는 구체적이고 민감한 방법을 설명합니다. 방법은 화학적으로 합성 표준에 비해 구조 분석을위한 glycosphingolipid 분자의 단계 - 현명한 조각을 허용 이온 트랩 질량 분광법을 활용합니다.

Glycosphingolipid 항원의 대량 Spectrometric 분석

출처: Hu, Y. et.al., 시험관 욕조 발효 시스템을 이용한 내도생제와 인간 장내 미생물군 간 상호작용 분석. J. Vis. 경험 (2019)이 영상은 외계다당류에 반응하여 인간 장내 미생물군이 단쇄 지방산 생성을 분석하는 과정을 보여줍니다. 이 연구는 가스 크로마토그래피를 이용한 미생물 발효물 분리 및 정량화를 강조합니다. 결과는 외다당류가 장내 숙주-병원체 역학에 영향을 미치는 미생물 대사산물을 조절한다는 것을 보여줍니다.

외계다당류에 대한 인간 장내 미생물 활성 조사

스포 도프 테라 리토 랄리스의 가트와 연관된 활성 박테리아 여행자는 pyrosequencing 기법에 결합 안정 동위체 프로빙 (SIP)에 의해 결정되었다. 이 방법을 사용하여, 지역 사회 내에서 대사 활성 박테리아 종의 식별은 높은 해상도와 정밀도로 이루어졌다.

제너럴의 장내 대사 활성 박테리아의 식별<em&gt; 스포 도프 테라 리토 랄</em&gt; DNA 안정 동위 원소를 통해 사용 프로빙<sup&gt; 13</sup&gt; C-포도당

효소 스크리닝을 위한 고처리량 분석에 대해 설명합니다. 즉시 사용 가능한 이 멀티플렉스 분석 키트는 사전 선택된 Chromogenic Polymer Hydrogel (CPH) 기질과 complex Insoluble Chromogenic Biomass (ICB) 기질로 구성됩니다. 표적 효소는 다당류를 분해하는 내효소(endo-enzymes)와 프로테아제(protease)입니다.

소설 불용성 발색 기질 분석 키트를 사용하여 탄수화물 분해 효소의 높은 처리량 검열

여기, 선물이 위장 장 관의 생리 조건을 시뮬레이션 bioreactors의 시리즈를 사용 하 여 콜론 생체 외에서, 의 본질적인 microbiota 경작을 위한 프로토콜.

인간의 본질적인 Microbiota 공부 기술을 경작 하는 체 외에서 고급 적용

우리 여기 높은 처리량 탐지 및 정량화 fucosylated 인간의 우유 oligosaccharides (Hmo)의 전체 셀 바이오 센서를 사용 하 여 설명 합니다. 또한 설명 여기, HMO 생산 종자의 분석으로이 플랫폼의 적응 신호 대 노이즈 비를 향상에 초점을 맞추고 합니다.

Fucosylated 인간 우유 Trisaccharides 유전자를 사용 하 여 바이오 컨텍스트에서 분석 인코딩된 바이오 센서

여기서 설명된 프로토콜은 시험관 내 배치 발효 시스템을 사용하여 내생생물학과 인간 장내 미생물 간의 상호작용을 조사하는 프로토콜이다.

체외 목욕 발효 시스템을 사용하여 내분비병제와 인간 장내 미생물 간의 상호 작용 분석

여기에 설명된 16S rRNA 메타게놈 시퀀싱 및 자유롭게 사용할 수 있는 도구를 사용하여 분석하는 미생물군유전체 프로파일링을 위한 간소화된 표준 작동 절차입니다. 이 프로토콜은 미생물군유전체 분야를 새로운 연구자뿐만 아니라 더 높은 해상도에서 박테리아 프로파일링을 달성하기 위한 방법에 대한 업데이트를 요구하는 연구자들에게 도움이 될 것입니다.

2단계 PCR 및 차세대 16S rRNA 유전자 시퀀싱을 사용한 미생물 분석

오픈 서닝은 이전에 알려지지 않은 글리칸 조성물로 장식된 글리코펩티드의 동정을 가능하게 한다. 이 기사에서는 Acinetobacter baumannii 를 모델로 사용하여 박테리아 샘플에 대해 공개 검색 및 후속 글리칸 중심의 글리코 펩티드 검색을 수행하기위한 간소화 된 접근 방식을 제시합니다.

Acinetobacter baumannii O 결합 글리코 펩타이드의 확인을위한 개방형 검색 기반 접근법의 적용

이 연구는 C-배당체를 절단할 수 있는 장내 세균을 효율적으로 스크리닝하고 분리하기 위한 일련의 표준 운영 절차(SOP)를 확립합니다.

C-배당체-절단 장내 세균의 스크리닝 및 분리

학습하세요 인간 장내 미생물총의 C-배당체 대사 효소를 구조 생물학 및 분석 화학 기법으로 특성화하는 방법을. 단백질 정제, 결정학, LC-MS/MS 및 NMR을 활용하여 효소 활성과 구조를 종합적으로 분석합니다.

인간의 장내 미생물총(microbiota)에서 C-배당체 대사 효소를 특성화하기 위한 구조 생물학 및 분석 화학 접근법

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