우리는 국립 보건원을 (1R21CA138373 감사하고이 작품의 재정 지원을위한 암 (SU2C)에 일어나. HY은을 위해 시드니 킴멀 재단에서 암 연구, 킴멀 학술 상을 미국의 협회에서 2009 Elion 수상에 감사합니다 암 연구 (SKF - 08-101) 및 국립 과학 재단 (National Science Foundation) 학부 조기 채용 수상 (NSF0954819).

1. 복제 고려 사항 <ol><li> NheI과 BamHI 제한 사이트와 5&#39; - phosphorylated 둘러싸인 관심 TMD를 대표하는 상업 준비 oligonucleotides는 pTox7 (직접 BamHI 제한 사이트 14 차례의 기본 쌍 삽입하여 연구실에서 수정) (그림 3) 순차적으로 소화에 출혈도 잡았 수 있습니다 BamHI과 NheI와 함께. 예제 oligonucleotide는 다음과 같습니다 : 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> TMD 순서는 12-24 잔류물 (짧은 시퀀스는 아마도 벡터 인코딩 소수성의 잔류물로 길쭉한합니다)해야합니다. 순차 잔여 삽입 및 ToxR에 TMD 상대의 회전에 수반하는 잔류 삭제 결과가. 15,16가 마지막으로 arabinose 농도가 0.001와 0.01 % 사이에서 다양 있어야 할 곳에 인터페이스를 조사하기 위해, TMD 디자인 네 가지 변종이 만들어되어야합니다 (W / V) 다른 TMD 시퀀스 사이의 β - galactosidase 신호의 최대 차이가 관찰되는 농도를 확인하기 위해, 다양한 표현 수준을 테스트하는 것은 다른 동질성이 가장 구분할 수있는 아래의 조건을 확인하는 것이 좋습니다. arabinose와 항생제 이외에도, 0.4 MM의 IPTG는 여러 TMDS 간의 동질성의 차이를 개선하는 데 사용할 수 있습니다. ToxR 측정은 최소한 사통으로 작성된 수행해야합니다. 전체 절차는 서로 다른 플라스미드 변환 최소한 세 번 반복해야합니다. 2. 세균성 문화의 성장 <ol><li> 부드럽게 15 ML 문화 튜브에 얼음과 송금에 FHK12 유능한 세포를 (200 μl) 해동. 플라스미드 DNA (200 NG)를 추가하고 30 분 얼음에 세포를 품어. </li><li> 42 90 s에 대한 부화하여 열 충격 세포 ° 2 분 얼음 부화 다음 C. </li><li> SOC 미디어 (800 μl) 추가하고 37 샘플을 품어 ° C 하나 H.위한 (300 RPM) 잡고 </li><li> 세중의 15 ML 문화 튜브의 변형 혼합물 50 μl와 chloramphenicol (30 μg / ML)와 arabinose (0.0025 % W / V) 5 ML의 LB 미디어의 예방. 37 샘플을 품어 ° C를 20 H.위한 (300 RPM) 잡고 (또는 문화의 5 μl은 96 - 웰 플레이트에 중간 100 μl를 예방하는 데 사용할 수 있습니다. 샘플의 큰 숫자를 처리 할 때이 방법은 유용합니다, 오류가 약간 높을 수 있지만. 이어질 것이 증발을 방지하기 위해 오류 매체와 가장 바깥쪽 우물을 채우기지만, 샘플을 사용하지 않는합니다. 마지막) 뚜껑과 parafilm와 접시 사이에 공동을 두 번 포장. </li></ol> 3. β - galactosidase 활동의 측정 <ol><li> 앞서 열을 가하다 판 - 리더 28 ° C. </li><li> 상단 (수성) 계층만을 차지해야하고, 대형 피펫 팁있는 저수지로 Z - 버퍼를 전송합니다. 96 - 웰 플레이트의 우물에 신선한 Z-buffer/chloroform 100 μl을 전송합니다. 사통으로 작성된 접시의 우물에 각 문화의 5 μl을 전송합니다. 빈 될 것입니다 푸르 웰스의 문화를 생략하십시오. </li><li> 세포 밀도를 결정하기 위해 접시의 OD를 측정 595. </li><li> 접시의 모든 우물을 Z-buffer/SDS 50 μl를 추가합니다. 세포를 lyze 10 분 접시 - Reader에서 접시를 흔들어. 셀 suspensions이 용해 후 명확한지, 그리고 필요한 경우 흔들림이 단계를 반복 있는지 확인하십시오. 불완전 용해는 Z-buffer/chloroform가 신선한 아니었 제안합니다. </li><li> 갓 모든 우물을 Z-buffer/ONPG 준비 50 μl를 추가, 20 분 매 30의 플레이트 리더와 측정 OD 405에 접시를 반환합니다. </li><li> 다음 방정식을 (를 비워 뺄 기억)를 사용하여 β - galactosidase 활동을 계산합니다. OD 405 / 분의 비율은 선형 모델 적합을 사용하여 1.0 OD 405 범위 0.0의 모든 데이터 요소를 사용하여 계산해야합니다. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="방정식 1" /> 다른 일에 기록된 때 밀러 단위 때로는 다릅니다. 그러므로, GPA와 같은 참조 구조는 각 시험에서 측정해야합니다. 그 값은 ToxR 값의 정상화를 위해 사용할 수 있습니다. </li></ol> 4. 단백질 표현을위한 컨트롤 <ol><li> 서양은 구조 사이에도 단백질 발현을 확인하기 위해 모래 바닥 수행합니다. microcentrifuge에 세중의 문화와 원심 분리기 50 μl (2000 RPM, 4 분)을 결합합니다. pipetting하여 표면에 뜨는를 제거하고 2 X 샘플 로딩 버퍼에 잔류 펠렛을 resuspend. </li><li> 표준 8 % 겔에서 7.5 μl를로드 1 시간당 5 분 125 V에서 electrophoreses를 수행합니다. 전송 후, 안티 MBP HRP - 복합 항체와 부화 및 시각화, 키메라 단백질은 때로는 48 KDA 주위에 본 몇 가지 열화 제품과 약 70 KDA에서 관찰됩니다. 내생 MBP도 (그림 5 참조) 45 KDA에서 관찰됩니다. </li></ol>5. 적절한 막 삽입에 대한 제어 말토오스 결합 단백질에 부족한 세포 라인은 키메라 TMD 구조의 적절한 막 삽입을 평가하는 데 사용됩니다. 유일한 탄소 소스로 말토오스 최소한의 미디어에 성장하는 경우에만 세포가 제대로 periplasm에있는 말토오스 결합 단백질과 막 - 필수 표현 제품을 표현이 성장 할 수 있습니다. <ol><li> PD28 세포를합니다 (FHK12 세포에 대한 설명) 변환 및 LB 매체 2 ML의 예방. 37 세포를 성장 ° C를 하루 (300 RPM) 잡고. </li><li> 펠렛은 3,500 rpm으로 원심 분리하여 세포를, 10 분, 4 ° C와 큰 팁 또는 부드러운 vortexing와 부드러운 pipetting하여 PBS (2 ML)에 resuspension로 씻으십시오. 펠렛 전지 (위)를, 두 번째 시간을 PBS로 세척 펠릿 그리고 마지막으로 PBS (1 ML)에 resuspend. </li><li> 37 세중의와 부화 5 ML 최소한의 미디어를 예방 resuspended 세포의 25 μl를 사용 ° C를 (300 RPM) 잡고. 15-25 H, 접시 - 판독기를 사용하여 96 - 웰 플레이트와 읽기에 각 시료 200 μl를 전송로 약 2 시간마다 사이 OD 595 수치를 가져가라. </li></ol> 6. 대표 결과 : 그림 4에 transmembrane 도메인의 oligomerization의 성향을 분석 ToxR transcriptional 기자 분석의 사용 예. 이전부터 우리가 transmembrane 도메인의 oligomerization를 조사 multispanning 멤브레인 - 필수 단백질 잠재 막 단백질 - 1 ToxR 등 다양한 기술에 의해 (LMP - 1) 14 Transmembrane 도메인 오 (TM5)가 oligomerize에 강한 성향을 전시 표시되었다.; 이것은 긍정적인 제어 비교 높은 밀러 유닛, GPA, 잘 설립 dimerizing 순서에 의해 입증됩니다. TM5, D150A에 해로운 돌연변이 oligomerize에 순서의 능력을 감소시킵니다. LMP - 1 TM1 크게 oligomerize하지 않으며 단지 빈 아닌 변화 FHK12 세포의 신호 위에 매우 낮은 밀러 단위 신호를 전시하고 있습니다. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="그림 1" /> 그림 1. 카툰은 ToxR 기자 분석을 묘사한. Transmembrane 도메인 (TMD)는 ToxR와 LacZ의 전사 활성화의 dimerization에 oligomerization 결과를 구동. LacZ 유전자의 제품은, β - galactosidase는 oligomerize에 대한 TMD의 성향의 측정으로 계량하실 수 있습니다. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="그림 2" /> 그림 2. 빛을 흡수 종 O - nitrophenolate (ONP)의 생산에 β - galactosidase 결과에 의해 ONPG의 가수 분해 절단. <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="그림 3" /> 그림 3. pToxR7의 플라스미드지도. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="그림 4" /> 그림 4. 대표 ToxR transcriptional 기자 분석은 잠재된 막 단백질 - 1 transmembrane 도메인의 oligomerization의 성향을 분석. transmembrane 도메인 1 (TM1)는 단지 약한 상호 작용을 전시 반면 Transmembrane 도메인 5 (TM5)은 강하게 oligomerizes. TM5에서 돌연변이의 D150A 상당히 oligomerize하기 위해 능력을 감소시킵니다. GPA는 강한 dimerization에 대한 긍정적인 제어 순서로 포함되어 있습니다. 빈은 untransformed FHK12 세포를 나타냅니다. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="그림 5" /> 그림 5. 단백질 발현을위한 서양 얼룩. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="그림 6" /> 그림 6. periplasm에 올바른 막 삽입에 대한 제어 PD28 complementation 분석. 대조군은 말토오스 결합 단백질에 결함 구조를 나타냅니다.

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관심 없음 충돌 선언하지 않습니다.

ToxR transcriptional 기자 분석은 oligomerize 수있는 잠재력을 지닌 transmembrane 시퀀스를 식별하는 손쉬운 방법입니다. 상호 작용은 세균 내부 멤브레인 내에서 발생하기 때문에,이 분석은 멤브레인 - 모방 환경에서 공부하는 시스템의 유효성과 관련된 문제를 circumvents. 하나의 플라스미드에 여러 개의 TMDS의 복제가 쉽게 병렬로 할 수 있으며, 전체 분석이 96 - 웰 플레이트 형식으로 진행 될 수 감안할 때,?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> BamHI 제한 효소 </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen 효소는 다른 공급 업체보다 더 효율적으로 발견되었습니다 </td></tr><tr><td> NheI 제한 효소 </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen 효소는 다른 공급 업체보다 더 효율적으로 발견되었습니다 </td></tr><tr><td> 15 ML 문화 튜브 </td><td> 피셔 사이 언티픽 </td><td> 14-956 - 1J </td><td></td></tr><tr><td> SOC 미디어 </td><td> Teknova </td><td> S0225 </td><td> 적절한 볼륨까지 만들어 autoclaving 소독. </td></tr><tr><td> LB 미디어 </td><td> 시그마 - 알드리치 </td><td> L7275 </td><td> 적절한 볼륨까지 만들어 autoclaving 소독. </td></tr><tr><td> Chloramphenicol </td><td> 시그마 - 알드리치 </td><td> CO 3 78 </td><td> 에탄올 30 MG / ML의 주식 솔루션은 냉동고에 저장 </td></tr><tr><td> Arabinose </td><td> Fluka </td><td> 10,839 </td><td> 물에 2.5 %의 주식 솔루션 (W / V)는 냉동고에 저장 </td></tr><tr><td> 나 2 HPO 4 </td><td> 시그마 - 알드리치 </td><td> S9390 </td><td></td></tr><tr><td> 아니 2 PO 4 </td><td> 시그마 - 알드리치 </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt 화학 </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 0.7 H 2 O </td><td> 시그마 - 알드리치 </td><td> 63,138 </td><td></td></tr><tr><td> 나트륨 dodecylsulfate (SDS) </td><td> 시그마 - 알드리치 </td><td> L6026 </td><td></td></tr><tr><td> 2 - Nitrophenyl β - D - galactopyranoside (ONPG) </td><td> 시그마 - 알드리치 </td><td> 73,660 </td><td></td></tr><tr><td> Z - 버퍼 </td><td></td><td></td><td> 16.1 g 나 2 HPO 4 5.5g 아니 2 PO 4 0.75g KCl 0.246g MgSO 4 1리터, 산도 7.0까지 확인 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 ML β - 메르 캅 토 에탄올, 2 ML 클로로포름은 Z - 버퍼 20 ML까지합니다. 800 rpm으로 1 분 1 분, 원심 분리기에 대한 소용돌이. 각 접시 신선합니다. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 MG SDS는 10 ML Z - 버퍼에 용해 </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 10 ML Z - 버퍼 40 MG ONPG. 각 접시 신선한 확인 </td></tr><tr><td> β - 메르 캅 토 에탄올 </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> 안티 MBP 단클론 항체 (HRP 복합) </td><td> 코 </td><td> E8038S </td><td></td></tr><tr><td> 말토오스와 거의 미디어 </td><td></td><td></td><td> 1 X M9 소금, 0.4 %의 말토오스, 1 MG / ML 티아민, 2 MM MgSO 4 </td></tr><tr><td> 96 - 웰 플랫 바텀 플레이트 </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> 플레이트 - 리더 </td><td> 베크맨 쿨터 </td><td> DTX880 다중 검출기 </td><td></td></tr><tr><td> 열탕 목욕 </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> 배양기를 흔들어 </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

disproven되어 이후 단백질 transmembrane 도메인의 oversimplified 전망은 단순히 인지질의 bilayers의 앵커 오래되었습니다. 많은 경우에 막 - 스패닝 단백질 행동의 고도로 복잡한 메커니즘을 발전시켜 왔습니다. 막 단백질들의 구조와 기능을 조절 할 수있는 1-3 편도는 구조 transmembrane의 oligomers를 형성, 소수성의 나선의 직접적이고 구체적인 접촉하는 것입니다. 4,5 대부분 최근이 작업은 우선적으로 수용액 및 드라이브 단백질 협회. 6,7는 그럼에도 불구하고, 단백질의 transmembrane 도메인에서 분자 인식 연구가 계속 중 그 뒤에 lags하는 다른 분자간 부대 비교 멤브레인 환경에서 발견된 아미노산의 분포에 초점을했습니다 수용성 지역. 주요 장애물은 남아있다 : 놀라운 특이성과 transmembrane의 oligomerization 얻을 수있는 친화력에도 불구하고, 그들의 협회 8 직접 측정은 도전입니다. 통합 멤브레인 단백질 기능 연구에 적용된 전통 방법론은 시험에서 시퀀스의 고유 불용성에 의해 방해하실 수 있습니다. transmembrane 도메인을 나타내는 합성 펩티드를 공부에서 얻은 통찰력 Biophysical 유용한 구조적 통찰력을 제공할 수 있습니다. 그러나 세제 micellar이나 세포 세포막을 모방하는 이러한 연구에 사용되는 리포좀 시스템의 생물 학적 관련은 종종 의문이며 펩티드는 이러한 조건 하에서 원시와 같은 구조를 채택하고 그들의 기능 동작은 진정한 네이티브 막 내에서 동작의 모드를 반영하지 ? 천연 인지질의 bilayers의 transmembrane 시퀀스의 상호 작용을 연구하기 위해 Langosch 실험실 ToxR transcriptional 기자 assays을 개발했습니다. 관심사 9 transmembrane 도메인이 periplasm 및 ToxR에 위치 말토오스 결합 단백질과 키메라 단백질로 표현되는 보고서를 제공하는 oligomerization의 수준 (그림 1). 지난 10 년, 여러 다른 그룹 (예 : Engelman, DeGrado, 샤이)는 더욱 최적화하고 ToxR 기자 분석을 적용. 10-13 다양한 ToxR의 assays는 세포 점막에 단백질 단백질 상호 작용을 테스트하는 황금 표준이되었습니다. 우리는 여기에 주로 Langosch에 의해 개발된 프로토콜을 따르는 우리 연구실에서 수행 전형적인 실험 작업을 보여줍니다. 이것은 일반적으로 적용되는 방법은 E.에 transmembrane 도메인 자체 협회의 분석에 유용합니다 β - galactosidase 생산이 TMD oligomerization의 성향을 평가하는 데 사용됩니다 대장균. TMD 유도 dimerization시, ToxR은 β - galactosidase에 대한 LacZ 유전자의 최대 규제를 일으키는 CTX업자에 바인딩합니다. colorimetric 판독이 lyzed 세포 ONPG를 추가하여 얻을 수 있습니다. 빛을 흡수 종 O - nitrophenolate (ONP) (그림 2)의 생산에 β - galactosidase 결과에 의해 ONPG의 가수 분해 절단.

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

단일 패스 transmembrane 도메인의 oligomerization의 성향 (TMDS)을 평가하는 효율적인 절차를 설명합니다. ToxR에 융합 TMD 구성된 키메라 단백질은 E. 대장균 기자 스트레인에 표현됩니다. TMD 유도 oligomerization는 ToxR, 해독 및 기자 단백질의 생산, - galactosidase의 활성의 dimerization이 발생합니다.

ToxR 분석에 의해 결정 Transmembrane 도메인 Oligomerization의 성향

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

transmembrane-domain-oligomerization-propensity-determined-toxr

Research

JoVE Journal

Biology

15.1K 조회수.  University of Colorado at Boulder. 단일 패스 transmembrane 도메인의 oligomerization의 성향 (TMDS)을 평가하는 효율적인 절차를 설명합니다. ToxR에 융합 TMD 구성된 키메라 단백질은 E. 대장균 기자 스트레인에 표현됩니다. TMD 유도 oligomerization는 ToxR, 해독 및 기자 단백질의 생산, - galactosidase의 활성의 dimerization이 발생합니다.

비디오: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

연구

생물학

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

로 표현 낭포 성 섬유증 막 횡단 전도력 규칙 단백질의 정제<em&gt; 사카로 마이 세스 세레 비시 애</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

형광 기반의 칼슘 동원 분석에 의해 G 단백질 결합 수용체의 특성

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

살아있는 세포의 단백질 조각 보완 분석 (PCA)에 의해 게놈 전체 단백질 - 단백질 상호 작용 상영

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

횡단 단백질의 재구성, 현미경 및 패치 클램프 연구 거대한 단일 층 소포에 전압 게이트 이온 채널, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

인트라 근질 그물 자리 칼슘의 광학 매핑<sup&gt; 2 +</sup&gt;와 랑겐 돌프 - 관류 토끼 심장에 막 횡단 잠재력

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

대한 유전 및 생화학 적 접근<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 단백질 올리고머의 평가 다음 Ryanodine 수용체 사례 연구

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

소포체와 미토콘드리아의 상호 작용의 연구에 의해<em&gt; 현장에서</em&gt; 근접 내고 분석 고정 세포의

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...