이 작품은 NIH (RO1-NS090390)에 의해 지원 되었다. 우리는 또한 박사 조 안 Steffanfor 기술 지원 및이 분석 결과의 개발 하는 동안 토론을 감사 하 고 싶습니다.

동물 윤리 문-실험 실시 했다 관심과 건강의 국가 학회 및 기관 동물 관리 및 사용 위원회 (에 의해 승인 된 동물 연구 프로토콜의 사용의 실험실 동물에 대 한 가이드와 함께 따라 IACUC), 어바인가 주 대학에는 AAALAC 공인 기관. 모든 노력 동물 고통 최소화 되었다.
1입니다. 세포의 용 해 버퍼의 준비
<ol><li>세포의 용 해 버퍼 "수용 성" 준비 (10 mM Tris pH 7.4, 1% 트리톤 X 100, 150 mM NaCl, 10% 글리세롤)와 0.22 μ m 필터를 통해 살 균 필터.<ol>
<li>작업 "수용 성" 세포의 용 해 버퍼, N-Ethylmaleimide (NEM)를 측정 하 고 철저 하 게 100mm phenylmethysulfonyl flouride (PMSF)에 100% 녹아 분해 EtOH. 참고: 세포의 용 해 버퍼 억제제: (i) 20 m m N-ethylmaleimide (NEM); (ii) 0.2 밀리미터 PMSF; (iii) 1 m m 나트륨 orthovanadate (나3보4); (iv) 1 µ g/mL leupeptin; (v) 1 µ g/mL aprotinin; (vi) 20 m m 나트륨 불 화물 (NaF). 주의: 프로토콜에 걸쳐 NEM 작업할 때 얼굴에 마스크를 착용 하십시오. 세포의 용 해 버퍼 작업 해야 할 활성 저 해제 되도록 신선한.</li>
		<li>다음과 같은 순서로 protease 억제제와 수용 성 세포의 용 해 버퍼를 추가 억제제: 나트륨 불 화물 (NaF), 찬 세포의 용 해 버퍼, 나트륨 orthovanadate (나3보4), Aprotinin와 Leupeptin 볼륨까지.</li>
	</ol>
</li>
	<li>"불용 성" 세포의 용 해 버퍼에 대 한 "성" 세포의 용 해 버퍼의 2 개 mL를 20 %SDS 500 µ L을 추가 하 여 4 %SDS 보충. 실 온에 둡니다. 참고: SDS 4 ° C에서 보관 하는 때 밖으로 침전 한다 이 온도 실 온에서 보관 하십시오. 최종 세포의 용 해 버퍼 구성: 10 mM Tris (pH 7.4); 1% 트라이 톤 X-100. 150 mM NaCl; 10% 글리세롤; 4 %SDS (불용 성만)</li>
</ol>2. 수용 성/불용 성 분류 프로토콜
참고: 회로도 대 한 그림 1 을 참조 하십시오.
<ol><li>각 샘플에 대 한 튜브의 두 세트를 라벨: "성"과 "불용 성"</li>
	<li>얼음 처럼 차가운 작업 성 세포의 용 해 버퍼 protease 억제제 (1.1.1 참조) 조직에 보충의 X-µ L를 추가 합니다. 참고: X = 볼륨에 따라 조직 금액; 표 1을 참조 하십시오.</li>
	<li>마우스 뇌 조직 (즉,가)에 대 한 dounce 유리 1 mL douncer에 "불용 성"에 피펫으로 천천히 30 번 표시 튜브 (얼음에 계속). 참고: 조심 하지 거품 양식을 것입니다 하 고 불완전 한 균질을 얻을 수 있습니다 douncing, 시작 후 액체의 표면에 휴식 한다.</li>
	<li>셀 (즉, 헬러, HEK293T), 한 번 감기, 1 x PBS로 세포를 씻어. PBS를 제거 하 고 세포 세포의 용 해 버퍼의 최소 볼륨 적용할 셀 스 크레이 퍼와 리프트. 참고: 셀 라인, 위에 셀 했다 6 잘 플레이트 당 5 x 105 셀에 도금 및 confluency의 가까이에 성장. 하나는 잘 비교 마우스가 사용 된 볼륨입니다.</li>
	<li>전송 homogenate 또는 세포 lysate에 레이블이 "불용 성" 튜브와 얼음 1 시간 (시작 시간 마지막 관 처리 후)에 lyse. 참고: 세포 lysates 위해 헤어 셀 덩어리 부 화를 시작 하기 전에 여러 번 triturate. 거품의 형성을 하지 마십시오. 짧게 소용돌이 펄스 1 각 샘플 lysing 반쯤 s.</li>
	<li>샘플 20 분 15000 x g에서 4 ° C에서 원심</li>
	<li>"성" 분수 상쾌한을 복구 합니다.<ol>
<li>피펫으로와 제거,이 분수 오염으로 펠 릿/흐림 레이어를 방해 하지 않도록 주의 하십시오. "성"에 피펫으로 녹는 분수 표시 관. 얼음에 계속.</li>
	</ol>
</li>
	<li>각 세척; 후 5 분 동안 15000 x g에서 4 ° C에서 500 µ L의 세포의 용 해 버퍼 (x2) 및 원심 분리기로 펠 릿을 세척 그것은 흐린 레이어 중 일부에서 pipetted는 경우 괜 찮 아 요입니다.</li>
	<li>최종 세척 후 조직 펠 릿만을 떠나 모든 나머지 워시 버퍼를 제거 합니다. 참고:이 시점에서 불용 성 샘플 지금 지켜져야 한다 실내 온도에.</li>
	<li>X-µ L 세포의 용 해 버퍼 4 %SDS 보충 resuspend 펠 릿 (볼륨 펠 릿 크기에 따라 조정할 수 있습니다; 표 1참조).</li>
	<li>30 각 샘플을 sonicate 프로브 sonicator 실내 온도에서 s (125 와트, 20 kHz 펄스, 진폭 40%, 재료의 표참조).</li>
	<li>30 분;에 대 한 샘플 (압 연 종)를 삶아 짧게 원심 (15 s) 어떤 샘플을 잃지으로 6000 x g에서.</li>
	<li>DC (세제 호환) 단백질 분석 실험을 수행 ( 재료의 표참조) 또는 (샘플 세포 배양에 대 한 각각의 세포의 용 해 버퍼의 1/5 및 1/10 조직 lysates의 시작된 희석 권장) 단백질 농도 측정에 Lowry 단백질 분석 결과. 참고: 단백질 농도 높은 세제 농도 때문 DC 또는 리 분석 결과 사용 하 여 읽을 해야 합니다.</li>
	<li>Aliquot 샘플을 동결-해 동 문제를 피하기 위해 50 µ L 배치로. 참고: 프로토콜 일시 중지할 수 있습니다 여기 추가 생 화 확 적인 분석 이전 두 분수-20 ° c.에 저장 하 여</li>
</ol>3. 서쪽 오 점 및 정량화
<ol><li>수행 보고 서쪽 오 점 다른1,13 30 µ g의 샘플 버퍼 (염료, 감소 x 1 로드 x 1.6) 로드에 1:1 희석 합니다. 참고: 분수 당 단백질의 30 µ g 충분 하지만 최적 해상도 대 한 조정 될 수 있다. 3-8% 트리 아세테이트 폴 리 아크릴 아 미드 젤은 불용 성 부분을 위해 사용 되어야 한다. 4-12% 두번째-트리 스 젤은 수용 성 부분을 위해 권장 됩니다.</li>
	<li>짧게 5 분 원심 분리기에 대 한 샘플 (압 연 종)을 종 기 (15 s) 6000 x g 샘플 수집에서.</li>
	<li>감소 된 샘플에 대 한 제조 업체의 프로토콜에 따라 분수 SDS 페이지와 서쪽 오 점 분석. 참고: 불용 성 분수 0.45 μ M 니트로 막에 전송 하는 경우 가장 좋은 해결 합니다. 관심사의 단백질의 크기에 따라 수용 성 부분을 위해 0.45 또는 0.2 µ M 니트로 사용할 수 있습니다. 일부 최적화 HMW 재료의 최고의 전송 하기 위해 필요할 수 있습니다.</li>
	<li>단백질 효율 로드를 시각화 하기 위해 전체 단백질 얼룩 (예를 들어, MEMcode, 참조 테이블의 재료)와 막 얼룩. 참고: 서쪽 오 점 평균 픽셀 강도 의해 분석할 수 있습니다. 녹는 분수 실험 시스템에 적합 한 최고의 집 유지 단백질에 정규화 될 수 있습니다. 상대 단백질 풍부도 불용 성 분수 가역 단백질 얼룩 또는 별도 오 점에 히스톤 3 정규화 하 여 확인 했습니다.</li>
</ol>

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저자는 공개 없다.

일관 되 고 양적 결과 보장 하기 위해 위의 프로토콜에 대 한 몇 가지 예방 조치 필요 합니다. 첫째, 두 분수에서 mHTT 저절로 형성 집계 여러 동결 해 동 주기, 시 시간이 지남에 특히 높은 농도에. 그것은 따라서 단백질 preps의 aliquots 고정만 위의 프로토콜에 설명 된 대로 시험을 실행 하기 전에 필요한 볼륨을 해 동 하는 중요 한. 또한, 불용 성 분수 녹고 시 흰색 비례적 수율, 경우 재구성 추가 5 s ?...

적절 한 접는 보장 하는 단백질 품질 관리 네트워크 장애 및 세포 단백질의 저하는 가능성이 Alzheimer의 질병 (광고), 파 킨 슨 병 (PD), 헌팅턴의 질병 (HD), 병 리 및 다른 "단백질 misfolding" 2,3장애. Proteostasis 네트워크 구성 요소와 병 리에 그들의 공헌에 대 한 자세한 이해는 그러므로 향상 된 치료 내정간섭을 개발 하는 중요 한. HD는 HD 유전자 huntingtin (HTT) 단백질4에서 polyglutamines (polyQ)의 확장 된 스트레칭 결과 내에서 CAG 반복의 비정상적인 확장에 의해 발생 합니다. HD pathogenesis의 일관 된 병 적인 기능이 이다는 결과 misfolding, 축적, 그리고 정상적인 세포 항상성 그리고 기능을 여러 가지 방법으로 방해를 표시 되었습니다 HTT 두뇌의 영역에서와 주변 조직에의 5 , 6. 동안 HTT 표현 편, 고가에 중간 가시 뉴런 선택적으로 취약 하 고 가장 명백 하 게 영향을 받는 주목할 만한 대뇌 피 질의 위축 또한 HD pathogenesis 연관.
HD 환자 및 동물 모델의 두뇌에 HTT의 집계의 형성 일반적으로 질병의 진행 및 지배적인 부정 이득의 mHTT7기능에 대 한 프록시 표식으로 봉사 했다. 비록 정확한 기계 장치는 단백질에 의해 misfolding와 집계 mHTT 유발 독성에 기여할 수 있습니다, 불분명 남아 있다 HD 환자와 다양 한 동물 모델의 뇌에서 이러한 흠도의 형성은 고정 하 고 피할 수 없는 기능입니다. 포함 표시 대부분의 N 맨끝을 포함 하는 누적된 HTT 조각 구성 확대 polyQ, 그 베이스를 나타내는 전장 HTT의 대안 접합8,처리9 할 수는 HD. N-터미널 HTT 파편의 병 인에 중요 한 역할 수 빠르게 집계, nucleate, 고 가속 또는 집계 프로세스10전파 HTT의 pathologic 형태를 구성 수 있습니다.
그러나, 이러한 포함의 존재 HTT 유발 독성 또는 세포 죽음11와 반드시 연결 되지 않습니다. HTT 불용 성 집계를 포함12성 oligomeric 종 및 β-시트 소 통해 녹는 단위체에서 집계 과정을 제안 하고있다. 이러한 다양 한 단백질 종 표준 생 화 확 적인 분석 실험을 통해 해결 낮은 세제 세포의 용 해 버퍼에서 그들의 안정성과 표준 생 화 확 적인 분석 실험을 사용 하 여 시각화에 어려움으로 인해 필드에 도전이 되었습니다. 따라서, 방법론 고려 사항 학위 및 누적 및 집계 mHTT의 패턴 감지에 중요 하다.
여기에 제시 된 프로토콜 제공 HTT, 특히 HD pathogenesis 및 질병 진행1,13 와 밀접 하 게 추적에 표시 되는 불용 성 HMW HTT 종 형성의 여러 중간체를 시각화 하는 방법 14. 해결 하 고 추적 mHTT의 여러 종 수 연구원은 질병 병 인을 연구 하 고 그들의 변조 및 질병 병 인에 미치는 영향을 통해 잠재적인 치료 내정간섭을 평가 생화학 도구를 제공 합니다.

<table><tbody><tr><td>Sterile Filter</td><td>Millipore</td><td>SCGP505RE</td><td>Screw cap, sterile vaccum filter</td></tr><tr><td>1 mL Tissue Grinder, Dounce</td><td>Wheaton</td><td>357538</td><td></td></tr><tr><td>Sonicator</td><td>Qsonica</td><td>Model Q125</td><td></td></tr><tr><td>DC Protein Assay</td><td>Biorad</td><td>5000111</td><td>Comparable to Lowry assay</td></tr><tr><td>Tris 1M buffer solution</td><td>Alfa Aesar</td><td>J60636</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>NaCl 5M solution</td><td>Teknova</td><td>S0251</td><td></td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td></td></tr><tr><td>20% SDS solution</td><td>Teknova</td><td>S0295</td><td></td></tr><tr><td>N-ethylmaleimide</td><td>Sigma</td><td>E1271</td><td></td></tr><tr><td>Phenylmethylsulfony flouride</td><td>Sigma</td><td>P7626</td><td>create 100mM stock solution in 100%EtOH, store at 4&amp;deg;C</td></tr><tr><td>Sodium orthovanadate</td><td>Sigma</td><td>S6508</td><td>Create 0.5M stock solution in water</td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td>Create 10mg/ml stock solution in water</td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td>Create 10mg/ml stock solution in water</td></tr><tr><td>Sodium Fluoride</td><td>Sigma</td><td>S4504</td><td>Create 500mM stock solution in water</td></tr><tr><td>Anti-HTT</td><td>Millipore</td><td>MAB5492</td><td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td></tr><tr><td>Anti-GAPDH</td><td>Novus Biologicals</td><td>NB100-56875</td><td>Use 1:1000 for soluble western blot</td></tr></tbody></table>

fractionation for resolution of soluble and insoluble huntingtin species

Misfolded 단백질의 축적 중심 Huntington의 질병 (HD)에 병 리 그리고 많은 다른 신경 퇴행 성 질환입니다. 특히, HD의 주요 병 적인 특징은 높은 분자량 복합물으로 돌연변이 체 HTT (mHTT) 단백질의 탈 선 축적 하 고 세포내 포함 시체 조각 및 다른 단백질으로 구성. 기존의 방법 측정 하 고 이해 하는 다양 한 형태의 mHTT 포함 된 집계의 기여 등 형광 현미경 검사 법, 서쪽 오 점 분석, 필터 트랩 분석 실험.
그러나, 이러한 방법의 대부분 특정, 구조 이며 따라서 mHTT 단백질 유량 집계 용 해도 및 해상도의 복잡 한 특성의 전체 상태를 확인 하지 않을 수 있습니다.
집계 mHTT 및 다양 한 수정된 양식 및 단지의 식별, 분리 및 세포 집계 및 파편의 가용 화는 필수입니다. 여기 우리 녹는 mHTT, 단위체, 올리고, 조각, 시각화를 격리 하는 방법을 설명 하 고 있는 불용 성 고 분자량 (HMW) 축적 mHTT 종. HMW mHTT 추적 질병 진행, 마우스 동작 표시기와 해당 하며 특정 치료 개입1군인이 변조 되었습니다. 이 방법은 마우스 뇌, 주변 조직 및 세포 배양 함께 사용할 수 있지만 다른 모델 시스템 또는 질병 상황에 적용할 수 있습니다.

분류에 따라 가용성과 불용 성 세포 lysates의 해상도 서양 분석 및 필터 지체 분석 (그림 2)를 사용 하 여 검색할 수 있습니다. 예를 들어, HEK293T 세포 transfection 시 약 (예를 들어, lipofectamine 2000)를 사용 하 여 페 했다, HTT exon과 97 글루타민을 포함의 인코딩 cDNA 1 개15 폴 리 프롤린 풍부한 지역 이어서 반복 하 고 이러한 셀 수 있었...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

메서드는 마우스 두뇌와 셀 문화에서 불용 해 성과 녹는 돌연변이 huntingtin 종의 분류에 대 한 설명 되어 있습니다. 설명 하는 메서드는 특성화 및 정량화 huntingtin 단백질 플럭스와 단백질 항상성 질병 병 인에 고 섭의 존재를 분석 에이즈의

가용성과 불용 성 Huntingtin의 해상도 대 한 분류

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

fractionation-for-resolution-soluble-insoluble-huntingtin

Research

JoVE Journal

Biochemistry

9.5K 조회수.  University of California Irvine. 메서드는 마우스 두뇌와 셀 문화에서 불용 해 성과 녹는 돌연변이 huntingtin 종의 분류에 대 한 설명 되어 있습니다. 설명 하는 메서드는 특성화 및 정량화 huntingtin 단백질 플럭스와 단백질 항상성 질병 병 인에 고 섭의 존재를 분석 에이즈의

비디오: Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

구조 연구 생산 이황화 - 안정화 Transmembrane의 펩타이드 단지

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

연구

화학

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

뇌막 분획 화 :<em&gt; Ex Vivo</em&gt; Subsynaptic Protein Localization 평가 방법

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

생화학

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

인간의 사후 뇌에서 세제 불용 성 단백질의 농축

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

효율적인 Refolding 및 Chemoreceptor Ligand 바인딩 도메인의 정화 방법

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

네이티브, 태그 Huntingtin Exon1 모노 머와 스모 융합 전략을 사용 하 여 소의 생성

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetotate 하이드로 아세테이트 도메인 함유 단백질의 발현, 정제, 결정화 및 효소 어세포

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

일시적 발현 시스템을 사용한 포유류 세포에서 전장 인간 헌팅틴 변이체의 효율적이고 확장 가능한 생산

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

돼지 신장과 마우스 간에서 FAHD1 단백질의 추출 및 정제

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.
Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.
The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

마우스 조직에서 안정적인 미세소관, 불안정한 미세소관 및 유리 튜불린을 분리하기 위한 정량적 미세소관 분획 기술

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

진핵 세포에서 번역 단지의 신속한 생체 내 고정 및 격리

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...