이 작품은 NIH (AI119813), 아칸소 대학에서에서 시작 및 아칸소 생명과학 연구소에서 수상에 의해 지원 되었다.

1. 사이트 감독 Mutagenesis 대상 유전자의
참고: MDH T7 발기인 CloDF13 원본 및 복사본 수가 20 ~ 4034 pCDF 1 벡터에서 아래 표시 됩니다.
<ol><li>뇌관에 의해 유전자에 140 위치에서 황색 정지 codon를 소개 (뇌관을 앞으로: GGTGTTTATGAC태그AACAAACTGTTCGGCG 및 뇌관을 역방향: GGCTTTTTTCAGCACTTCAGCAGCAATTGC)의 사이트 지시 된 mutagenesis 키트 명령 다음.</li>
	<li>야생-타입 malate dehydratase의 유전자를 포함 하는 템플릿 플라스 미드를 증폭 하 고 중 합 효소 연쇄 반응 (PCR) 반응에 의해 정지 codon 돌연변이 삽입 합니다. 반응 혼합물에서 포함 2 X DNA 중 합 효소 효소 혼합의 12.5 µ L, 10 µ M 앞으로 뇌관, 10 µ M 역 뇌관의 1.25 µ L, 템플릿 DNA의 1 µ L의 1.25 µ L (20 ng / µ L) (pCDF 1 플라스 미드의 야생-타입 MDH 유전자를 포함), 그리고 9 µ L nuclease 무료 물.<ol>
<li>PCR 반응 매개 변수를 사용 하 여 다음과 같습니다: 30 98 ° C에 변성을 초기 s; 10의 25 주기 98 ° C, 30에서 s s 55 ° C, 및 72 ° C에서 3 분에서 3 분 동안 72 ° C에서 최종 확장. PCR, 후 circularization 및 서식 파일 제거에 대 한 실 온에서 1 h 킷에서 증폭 된 자료 니 리가 DpnI 효소 혼합에 직접 추가 합니다. 참고: 반응 혼합물의 PCR 제품, 반응 버퍼 X 2의 5 µ L, 10 X 키-리가-DpnI 효소 혼합, 1 µ L nuclease 무료 물 3 µ L 1 µ L를 포함합니다.</li>
	</ol>
</li>
	<li>5 µ L 25 µ L의 튜브에 반응 혼합의 유능한 대장균 해 동 추가 키트에서 DH5α 셀. 신중 하 게 혼합, 튜브를 터치 하 고 30 분 열 충격 30 s, 및 추가 5 분 동안 얼음에 42 ° C에 혼합물에 대 한 얼음에 혼합물을 놓습니다.<ol>
<li>혼합물으로 실내 온도 Catabolite 억압 (SOC) 미디어 키트에서와 함께 슈퍼 최적의 국물의 600 µ L를 플라스틱, 250rpm에서 떨고 함께 60 분 동안 37 ° C에서 품 어, 해당 항생제와 lysogeny (파운드)의 국물 한 접시에 100 µ L를 확산 그리고 250 rpm에서 떨고와 37 ° C에서 밤새 품 어.</li>
	</ol>
</li>
	<li>6 mL 신선한 파운드 미디어 해당 항생제로 4-6 한 식민지를 선택 하 고 250 rpm에서 떨고와 하룻밤 37 ° C에서 품 어. 다음 제조 업체의 설명서, 다음 올바른 위치에 정지 codon 돌연변이 확인 하는 서비스 공급자의 프로토콜에 따라 DNA 연속을 위한 송신 플라스 미드 플라스 미드 정화 키트에 의해 각 숙박 문화에서 플라스 미드를 추출 합니다.</li>
	<li>1 mL 하룻밤 문화와 300 µ L 100 %DMSO 혼합 하 여 스트레인-80 ° C에서 올바른 시퀀스를 저장 합니다.</li>
</ol>2입니다. 표현의 Acetylated 단백질
<ol><li>최적화 된 acetyllysyl-tRNA 합성33 의 유전자를 삽입 하 고 최적화 된 tRNAPyl 32 pTech 플라스 미드로. 제정 lpp 발기인에서 tRNA 합성 유전자를 놓습니다. 제정 proK 발기인34에서 tRNA 유전자를 놓습니다.<ol>
<li>공동 변환 식 벡터34 malate 효소의 돌연변이 태그 포함 된 유전자를 포함 하 고 최적화 된 acetyllysine 설립 시스템을 은닉 하는 플라스 미드 25 µ L로 해 동된 유능한 대장균 BL21(DE3) 세포에 의해 10 s, 및 추가 5 분 동안 얼음에 42 ° C에서 열 충격.</li>
		<li>혼합물으로 실내 온도 SOC 미디어의 600 µ L를 플라스틱, 275 rpm 동요와 함께 60 분 동안 37 ° C에서 품 어, 100 µ g/mL 스와 50 µ g/mL 페니, 한 접시에 100 µ L를 확산 그리고 275 rpm에서 떨고와 37 ° C에서 밤새 품 어.</li>
	</ol>
</li>
	<li>접시에서 단 하나 식민지를 선택 하 고 15 mL 신선한 파운드 미디어 100 µ g/mL 스와 250 rpm의 속도로 흔들어 하룻밤 37 ° C에서 50 mL 튜브에 50 µ g/mL 페니로 예방. 1 L 플라스 크에 항생제와 300 mL 신선한 파운드 미디어 15 mL 숙박 문화를 전송 하 고 250 rpm에서 떨고와 37 ° C에서 품 어.</li>
	<li>Acetyllysine 100 m m 재고 솔루션, 4 ° c.에 저장 물으로 분해 흡 광도 600 0.5에 도달 하는 때 성장 매체를 5mm acetyllysine 및 20mm 니코틴 (deacetylases의 억제제)를 추가 nm.<ol>
<li>37 ° c, 250 rpm에서 떨고 추가 1 시간에 대 한 세포 성장 다음 0.5 m m IPTG 단백질 표정, 추가 하 고 180 rpm에서 떨고, 25 ° C에서 세포 성장. 참고: 식 조건이 다른 단백질에 대 한 최적화를 할 수 있습니다.</li>
	</ol>
</li>
	<li>15 분 동안 4 ° C에서 3000 x g에서 centrifuging 여 세포를 수집 하 고 상쾌한, 삭제 셀 인산 염 버퍼 식 염 수 (PBS) 버퍼 (10 m m 나2HPO4, 1.8 m m KH2포4, 137 m m NaCl, 2.7 m m KCl)와 펠 릿을 세척. 5 분 동안 4 ° C에서 10000 x g에서 씻어 세포를 수집, 상쾌한, 삭제 및 셀 알 약-80 ° c.에 저장</li>
</ol>3입니다. Acetylated 단백질의 정화
<ol><li>얼음, 냉동된 셀 펠 릿을 녹여 그리고 다시 (50 mM tris (hydroxymethyl) aminomethane (Tris) pH 7.8, 300 mM NaCl, 20mm 이미, 그리고 20mm 니코틴아미드), 세포의 용 해 버퍼의 15 mL와 β-mercaptoethanol와 1 µ L Benzonase nuclease (250 대)의 5 µ L 일시 중단.</li>
	<li>40 kHz 쥡니다 70% 파워 10 사이클 간격 30 다음 10 s 짧은 파열의 출력에 의해 세포를 휴식 양식 원유 추출에 대 한 s. 4 ° c.에 25 분 20000 x g에서 원심 분리기 원유 추출 상쾌한 0.45 μ 막 필터를 필터링 하 고 니켈 nitrilotriacetic 산 (Ni-NTA) 수 지 물과 세포의 용 해 버퍼의 20 mL 20 mL와 equilibrated의 1 mL를 포함 하는 열에 로드 합니다. 참고: 쥡니다 사용할 수 없는 경우에 셀 온화한 세제에 의해 깨진 수 있습니다.</li>
	<li>20 mL (50 mM Tris pH 7.8, 300 mM NaCl, 50mm 이미, 및 20 m m 니코틴아미드) 워시 버퍼의 열 세척 하 고의 차입 버퍼 (50 mM Tris pH 7.8, 300 mM NaCl, 150mm 이미, 및 20 m m 니코틴아미드) 2 mL와 함께 elute.</li>
	<li>제조업체의 설명서에 따라 PD-10 열 버퍼 (25mm Tris pH 7.8와 10 mM NaCl) 담와 차입 분수 desalt. Bradford 단백질 분석 실험 시 약의 지시에 따라 eluted 단백질의 농도 측정 합니다. Desalted 단백질은 추가 실험을 위한 준비 이다. 참고: 단백질의 50% 글리세롤 재고 확인 하 고 저장을 위한-80 ° C에서 유지.</li>
</ol>4. 생 화 확 적인 특성 Acetylated 단백질의
<ol><li>
SDS 페이지 및 질량 분석 분석입니다.
	<ol>
<li>나트륨 라우릴 황산 염 (SDS) 샘플 버퍼 (SDS 샘플 버퍼 X 2 µ L 4와 5 µ L 단백질 견본) 5 분 동안 105 ° C에서 2 mL 튜브와 단백질 변성, 2000 x g 10에서 혼합물을 원심 s, 4-20% 나트륨 라우릴 황산 polyacrylamide 젤 electroph에 로드 oresis (SDS-PAGE) 젤, 고 200 V 30 분 실행 합니다.</li>
		<li>증류수와 젤을 세척 하 고 3 번 과정을 반복 5 분 동안 부드럽게 흔들어. 물, 삭제 하 고 젤 부드러운 동요와 1 h Coomassie 파란 얼룩과 얼룩. 증류수와 젤 드 얼룩, 30 분, 그리고 반복이 드 3 번 얼룩에 부드럽게 흔들어.</li>
		<li>Coomassie 파란 얼룩이 SDS 페이지 젤에 33 kDa에 밴드를 잘라내어 질량 분석 시설 또는 회사는 acetyllysine 디자인 된 위치에서 통합 되었다 확인 하. 참고: 질량 분석 분석의 프로토콜은 이전 실험34을 따 랐 다.</li>
	</ol>
</li>
	<li>
서 부 럽
	<ol>
<li>4.1 단계에서 동일한 프로토콜 SDS 페이지 젤을 실행 합니다. 실행 젤, 후 15 분에 대 한 전송 버퍼 (25mm Tris, 192 m m 글리신, pH 8.3 및 20% 메탄올) 젤을 담근 다.</li>
		<li>0.2 µ m, 1 분에 대 한 메탄올을 가진 7 cm x 8.5 c m polyvinylidene difluoride (PVDF) 막 활성화 되 고 전송 샌드위치를 준비 하기 전에 전송 버퍼와 린스. 참고: 메탄올은 피부 접촉, 눈 접촉, 섭취, 또는 흡입 위험. 심한 노출은 죽음에 발생할 수 있습니다.</li>
		<li>음극에서 양극 (스폰지 필터 종이, SDS 페이지 젤, PVDF 막, 필터 종이, 스폰지)에 전송 샌드위치를 확인 합니다. 스택의 350의 정 전류에서 실행 전송 탱크에 넣어 45 분 mA. 참고: 전송 시간 최적화를 할 수 있습니다.</li>
		<li>씻어 25 mL Tris 버퍼 식 염 수, 0.1 %PVDF 막 트윈 20 (TBST) (137 m m NaCl, 20 mM Tris, 0.1% 트윈 20, pH 7.6) 부드러운 떨고와 5 분에 대 한 버퍼. 5% 소 혈 청 알 부 민 (BSA) 실 온에서 1 h TBST 버퍼에서와 막을 차단 합니다.</li>
		<li>HRP 활용 된 acetyllysine-항 체로 부드러운 떨고와 하룻밤 4 ° C에서 5% BSA TBST에 희석 1 µ g/mL의 최종 농도와 막 품 어. 참고: 빠른 결과 위해 1 시간 실 온에서이 단계를 수행 수 있습니다. 항 체의 희석 최적화를 할 수 있습니다.</li>
		<li>부드러운 진동으로 5 분 동안 20 mL TBST 버퍼와 막 씻어 4 번 단계를 반복. 제조업체의 지침에 따라 화학 기질 막에 적용 합니다. 와 전 하 결합 소자 (CCD) 카메라 기반 영상 신호를 캡처.</li>
	</ol>
</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+protein+acetylation:+new+discoveries+unanswered+questions.">Bacterial protein acetylation: new discoveries unanswered questions.</a> Curr Genet. 62 (2), 335-341 (2016).</li><li>Albaugh, B. N., Arnold, K. M., Lee, S., Denu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoacetylation+of+the+histone+acetyltransferase+Rtt109.">Autoacetylation of the histone acetyltransferase Rtt109.</a> J Biol Chem. 286 (28), 24694-24701 (2011).</li><li>Neumann, H., Peak-Chew, S. Y., Chin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoding+N&#949;-acetyllysine+in+recombinant+proteins.">Genetically encoding N&#949;-acetyllysine in recombinant proteins.</a> Nat chem biol. 4 (4), 232-234 (2008).</li><li>Fan, C., Xiong, H., Reynolds, N. 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저자는 공개 없다.

비정규 아미노산 (ncAAs)의 유전 결합 ncAA 청구 tRNA에 의해 할당 된 codon, 주로는 황색 정지 codon UAG36,37,38,39의 억제에 기반 해당 anticodon 포함 하. UAG codon 릴리스 요소-1 (RF1) 박테리아에 의해 인식 된다로 알려져 있다 그리고 그것은 또한 억제할 수 있습니다 의해 정식 아미노산 (cAAs)로 청구 하는 호스트에?...

포스트 번역 상 수정 (PTMs) 단백질의 번역 과정 후 발생 하 고 공유 추가 기능 그룹에서 발생 하는 아미노산 잔류물, 유전자를 포함 하 여 거의 모든 생물 학적 과정에 중요 한 역할을 재생을 전사, 스트레스 반응, 세포 분화, 및 물질 대사1,2,3 날짜 하려면, 약 400 독특한 PTMs 확인된4되었습니다. 게놈과는 프로테옴의 intricacy는 단백질 PTMs로 대부분 증폭 단백질 활동 및 지역화, 고 단백질, 핵 산, 지질, 및 공동 인자5등 다른 분자와 상호 작용에 영향을 미칠.
단백질 acetylation 지난 2 년간6,7,,89,10,11,12PTMs 연구의 최전선에 왔다. Lysine acetylation 처음 발견 되었다 히스톤에 이상의 50 년 전13,14은 잘 조사 되어, 80 개 이상의 녹음 방송 요인, 레 귤 레이 터, 및 다양 한 단백질15에 알려져 있다 16,17. 단백질 acetylation에 대 한 연구 뿐만 아니라, 규제 메커니즘의 깊은 이해를 제공 하지만 다양 한 역 기능 acetylation18,19, 로 인 한 질병에 대 한 치료 가이드 20 , 21 , 22 , 23. lysine acetylation 진핵생물에서 발생 하지만 최근 연구는 단백질 acetylation 또한 세균 생리학, chemotaxis, 내 산 성, 활성화 및 안정화를 포함 하 여 주요 역할 재생 믿어 pathogenicity 섬과 다른 독성 단백질24,25,26,27,,2829관련.
Lysine acetylation 화학적 특성을 일반적으로 사용 되는 방법은 사이트 지시 된 mutagenesis 사용 하는. 글루타민은 비슷한 크기와 극성 때문에 acetyllysine의 모방으로 사용 됩니다. 아르기닌은 생리 적인 조건 하에서 그것의 긍정적인 요금을 유지 하지만 acetylated 수 없습니다 비 acetylated lysine 모방으로 활용 됩니다. 그러나, 두 모방 하지 진짜 isosteres 이며 항상 예상된 결과30를 생성 하지 않습니다. 가장 엄격한 접근은 어렵거나 lysine acetylation 자연7,11의 낮은 산출할 인해 가장 고전적인 방법에 대 한 특정 리 진 잔류물에 균질 acetylated 단백질을 생성 하는 것입니다. 이 문제는 설계 pyrrolysyl-tRNA 합성을 사용 하 여 유전자 코드 확장 전략에 의해 unraveled 되었습니다 tRNA 충전 Methanosarcinaceae 종에서 벗어나 acetyllysine와Pyl 활용 호스트 변환 기계는 UAG를 억제 하는 mRNA에 codon를 중지 하 고 대상 단백질31의 설계 위치에 acetyllysine의 지휘. 최근에, 우리는 향상 된 EF-부 엉-바인딩 tRNA32 와 업그레이드 된 acetyllysyl-tRNA 합성33이 시스템을 최적화 했습니다. 또한, 우리는 malate 효소34 와 tyrosyl-tRNA 합성35의 acetylation 연구에서이 향상 된 통합 시스템을 적용 했습니다. 여기, 우리가 우리가 광범위 하 게 실증 사례로 공부 malate 효소 (MDH)를 사용 하 여 생화학 식별 분자 클로닝에서 순전히 acetylated 단백질을 생성 하기 위한 프로토콜을 보여 줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bradford protein assay</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">5000006</td>
			<td style="width:167px;">Protein concentration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4x Laemmli Sample Buffer</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610747</td>
			<td style="width:167px;">SDS sample buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Coomassie G-250 Stain</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">1610786</td>
			<td style="width:167px;">SDS-PAGE gel staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">4-20% SDS-PAGE ready gel</td>
			<td style="width:113px;">Bio-Rad</td>
			<td style="width:119px;">4561093</td>
			<td style="width:167px;">Protein determination</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ac-K-100 (HRP Conjugate)</td>
			<td style="width:113px;">Cell Signaling</td>
			<td style="width:119px;">6952</td>
			<td style="width:167px;">Antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IPTG</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">194</td>
			<td style="width:167px;">Expression inducer</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">N&#949;-Acetyl-L-lysine</td>
			<td style="width:113px;">CHEM-IMPEX</td>
			<td style="width:119px;">5364</td>
			<td style="width:167px;">Noncanonical amino acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PD-10 desalting column</td>
			<td style="width:113px;">GE Healthcare</td>
			<td style="width:119px;">17085101</td>
			<td style="width:167px;">Desalting</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">E0554</td>
			<td style="width:167px;">Introducing the stop codon</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BL21 (DE3) cells</td>
			<td style="width:113px;">NEB</td>
			<td style="width:119px;">C2527</td>
			<td style="width:167px;">Expressing strain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">27106</td>
			<td style="width:167px;">Extracting plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Ni-NTA resin</td>
			<td style="width:113px;">QIAGEN</td>
			<td style="width:119px;">30210</td>
			<td style="width:167px;">Affinity purification resin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">nicotinamide</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3376</td>
			<td style="width:167px;">Deacetylase inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">&#946;-Mercaptoethanol</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6250</td>
			<td style="width:167px;">Reducing agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BugBuster Protein Extraction Reagent</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">70584</td>
			<td style="width:167px;">Breaking cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Benzonase nuclease</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1014</td>
			<td style="width:167px;">DNase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ECL Western Blotting Substrate</td>
			<td style="width:113px;">ThermoFisher</td>
			<td style="width:119px;">32106</td>
			<td style="width:167px;">Chemiluminescence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Premixed LB Broth</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97064</td>
			<td style="width:167px;">Cell growth medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine serum albumin</td>
			<td style="width:113px;">VWR</td>
			<td style="width:119px;">97061-416</td>
			<td style="width:167px;">western blots blocking</td>
		</tr>
	</tbody>
</table>

a facile protocol to generate site-specifically acetylated proteins in escherichia coli

포스트 번역 상 수정 단백질의 특정 위치에서 발생 하는 다양 한 세포질 프로세스에서에서 중요 한 역할을 보여왔다. 그 중, 가역 lysine acetylation 생활의 모든 영역에서 가장 넓게 분배 된 중 하나입니다. 수많은 acetylome 질량 분석 기반 연구를 수행 하는 있지만 더이 상 상속 acetylation 대상의 제한 되었습니다. 가능한 이유 하나 대부분 클래식 생 화 확 적인 방법에 의해 원하는 위치에 순전히 acetylated 단백질을 생성 하기 어렵습니다. 이 문제를 해결 하려면 유전자 코드 확장 기술 cotranslational 설립을 직접 설계 pyrrolysyl-tRNA 합성 변종, Methanosarcinaceae 종에서 그것의 동족 tRNA의 쌍을 사용 하 여 적용 된 관심사의 단백질에 특정 사이트에 acetyllysine의. Histone acetylation의 연구에서 첫 번째 응용 프로그램, 후이 이렇게 다양 한 단백질에 acetylation 연구 촉진 했다. 이 작품에서는, 우리는 호스트 모델 박테리아 대장균 을 사용 하 여 site-specifically acetylated 단백질을 생산 하는 손쉬운 프로토콜을 증명 하고있다. Malate 효소는이 작품에서 데모 예제로 사용 되었습니다.

야생-타입 MDH 그건 1 L 문화 당 31 mg acetylated MDH 단백질의 수익률 1 L 문화, 당 15 밀리 그램 이었다. 그림 1에서 보듯이 순화 된 단백질 SDS-PAGE로 분석 되었다. 야생-타입 MDH 긍정적인 제어34로 사용 되었다. 성장 매체에서 acetyllysine (AcK) 설립 시스템 및 돌연변이 mdh 유전자를 은닉 하는 세포에서 하지만 AcK 없이 순화 하는 단백질은 ...

Watch this Scientific Journal Video about A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli at JoVE.com

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

유전자 코드 확장 acetylation 단백질을 포함 한 생물 학적 과정의 넓은 범위를 공부 하는 강력한 도구 역할을 합니다. 여기 균질 생성 하기 위한이 기술을 악용 하는 손쉬운 프로토콜 acetylated 단백질 대장균 세포에 특정 사이트에 설명 합니다.

Site-Specifically 생성 하는 손쉬운 프로토콜 Acetylated 대장균 에서 단백질

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

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Immunology and Infection

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

6.8K 조회수.  University of Arkansas. 유전자 코드 확장 acetylation 단백질을 포함 한 생물 학적 과정의 넓은 범위를 공부 하는 강력한 도구 역할을 합니다. 여기 균질 생성 하기 위한이 기술을 악용 하는 손쉬운 프로토콜 acetylated 단백질 대장균 세포에 특정 사이트에 설명 합니다.

비디오: A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

시각화 및 정량하기 위해 형광 단백질을 사용하여<em&gt; 클라미디아</em&gt; 액포 성장 역학 살아있는 세포에서

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

연구

면역학 및 감염병학

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

감염 프로토콜<em&gt; 예쁜 꼬마 선충</em&gt;와<em&gt; 살모넬라 티피 뮤 리움 (Salmonella typhimurium)</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic의 비 침습 모델<em&gt; 대장균</em&gt; 신생아 쥐에 감염

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

한 단계의 부정적인 크로마토 그래피 정제<em&gt; 헬리코박터 파이로리</em&gt; 호중구 - 활성화 단백질 과발현<em&gt; 대장균</em&gt; 배치 모드에서

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

형태 변화를 규명하기위한 프로토콜<em&gt; 클로스 트리 디움 디피 실</em&gt; 항생제 치료에 대한 반응

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

밀도 Transposon 삽입 라이브러리를 사용 하 여 세균성 활용 Enterobacterial 종자와 같은 대장균 또는 Shigella flexneri 의 창조

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Site-Specifically 생성 하는 손쉬운 프로토콜 Acetylated 대장균 에서 단백질

요약

더 많은 비디오 탐색

이 비디오의 챕터

시각화 및 정량하기 위해 형광 단백질을 사용하여

감염 프로토콜

Neuropathogenic의 비 침습 모델

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

한 단계의 부정적인 크로마토 그래피 정제

형태 변화를 규명하기위한 프로토콜

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

밀도 Transposon 삽입 라이브러리를 사용 하 여 세균성 활용 Enterobacterial 종자와 같은 대장균 또는 Shigella flexneri 의 창조

인간의 과발현과 정제

녹색 형광 단백질 표현 하는 향상 된 사용 하 여 대장균 마우스 복 막 대 식 세포 식 균 작용을 평가 하기 위해