이 작품은 건강 연구의 캐나다 연구소에 의해 지원되었다. CSC는 자연 과학 및 캐나다의 공학 연구위원회에서 박사 친목의 지원을받는되었다. FD는 등급 II 캐나다 연구 의자입니다.

<html> 전체 Reconstitution 프로세스 reconstitution 과정은 세제 - solubilized phospholipids의 존재에 정화 MalFGK이 컴플렉스로 ​​막 비계 단백질 (MSP)를 혼합하여 시작합니다. 단계는 바이오 비즈 나 Amberlite (그림 1)라는 흡착제 폴리스티렌 소재로 세제의 느린 제거을합니다. 자동 조립 공정 때문에 소수성 phospholipids, MalFGK이 복잡하고 MSP amphipathic 단백질의 표면 사이의 apolar 상호 작용의 가능성이 가장 높은 발생합니다. 최종 제품은 MalFGK이 복잡한 주변 MSP 포장의 두 분자로 만든 원반 모양의 입자입니다. 입자는 초 원심 분리 및 분석 크기 배제 크로마토 그래피에 의한 알데히드 부가 물 및 집계에서 구분됩니다. 입자는 기본 - 겔 전기 영동 및 동적 광 산란 분광법을 특징으로하고 있습니다. 막의 제목 &quot;&gt; 1. 준비 비계 단백질, MSP <ol><li> 자신의 태그 MSP는 (버전 MSP1D1 1) E.의 플라스미드 pMSP1D1에서 생산됩니다 대장균 37 3 시간 0.5 MM 이소 프로필 β-D-1-thiogalactopyranoside와 OD 600 ~ 0.5에서 유도 BL21 (DE3) ° C. </li><li> 셀은 4시 10 분 ° C에 5,000 XG에서 원심 분리하여 수확, 100 μM의 phenylmethanesulfonyl 불화를 포함하는 TSG10 버퍼에 resuspended 있습니다. </li><li> 세포는 8,000 PSI에 프랑스 프레스 (3 배)으로 lysed하고 불용성 물질은 4 ° C.에서 10 분 동안 5,000 XG에서 원심 분리에 의해 제거됩니다 MSP를 포함하는 수용성 분율은 45 분에 125,000 XG에 초 원심 분리에 의해 분리된다. </li><li> MSP는 니켈 - 상기 킬레이트 화 크로마토 그래피는 TSG10 버퍼에 ~ 1.5 ML 니켈 세 파로스 HP를 사용하여 정화되어 있습니다. 오염 물질은 50 MM 이미 다졸을 포함하는 TSG10 버퍼로 씻어 수 있습니다. MSP는 600 MM를 포함하는 TSG10 버퍼로 용출되어이미 다졸. 정화 단백질은 TSG10 버퍼에 dialyzed와 ~ 10-15 MG / ML의 단백질 농도에서 -70 ° C에 저장됩니다. </li></ol> 2. MalFGK이 단지의 작성 <ol><li> MalFGK이 단지는 MalK의 C-말단에 자신의 태그, E.의 플라스미드 pBAD22 - FGK에서 표현됩니다 대장균 BL21 (DE3)와 37 3 시간에 0.2 % L-arabinose와 OD 600에서 ~ 0.5 유도 ° C. </li><li> 단계 1.3에서와 같이 세포 용해 및 원심 분리 후, 막 펠릿은 5 MG / ML의 최종 농도에 TSG20 버퍼에 resuspended 있습니다. 자료는 부드러운 진동과 4에 3 시간에 1 % W / V N-dodecyl-β-maltopyranoside (DDM) ° C로 solubilized 있습니다. </li><li> 불용성 물질은 단계 1.3에서와 같이 초 원심 분리에 의해 제거하고 표면에 뜨는는 solubilized을 포함하고 있습니다 MalFGK이 단지는 수집 단계 1.4에서와 같이 정화하지만, 투석하지 않고 있습니다. </li><li> FurtheR 정화는 0.5 ML / 분의 유량에서 Superdex 200 HR 3백분의 10 열 TSGD 버퍼에 겔 여과 크로마토 그래피에 의해 달성된다. </li></ol> 3. Phospholipids의 작성 <ol><li> E. 대장균 총 지방질은 클로로포름에 용해 추출하는 것은 나사 캡 microfuge 튜브 1,000 nmol aliquots로 구분됩니다. 용매는 질소의 완만 한 흐름에 따라 증발 진공 건조기에서 추가 하룻밤 건조합니다. </li><li> 지질 필름은 5 nm의 최종 농도에서 TS 버퍼에 용해하고 힘차게 vortexed와 sonicated 수 있습니다. 용해 지질 인해 수성 TS 버퍼에서의 일반 불용성으로 약간 불투명 나타납니다,하지만 그들은 정지에 남아 있어야합니다. DDM은 해결책이 분명해진다되는 0.5 % (~ 10 ㎜)의 최종 농도에 추가됩니다. </li><li> 지질 혼합물은 물 목욕에 배치 및 펄스는 ~ 5 초 동안 5 회에 sonicated 수 있습니다. 지질 혼합물은 1 꼬마의 최대 4 ° C에 저장됩니다K. </li></ol> 4. 바이오 비즈의 작성 <ol><li> 약 10-15 ML은 (드라이 볼륨) 바이오 비즈는 50 ML 튜브에 배치됩니다. </li><li> 구슬이 연속 100 % 메탄올, 95 % 에탄올, milliQ H 2 O, 그리고 마지막으로 TS 버퍼 (두 번 각)의 50 ML에 세척되어 있습니다. </li><li> 세척 구슬은 4에 저장됩니다 ° C ~ 10 ML TS 버퍼 인치 </li></ol> 5. Nanodisc의 Reconstitution <ol><li> MSP는 ~ 7 MG / ML (~ 0.3 ㎜)의 최종 농도에 TSGD 버퍼에 희석되어 있습니다. </li><li> MSP : TSGD 버퍼에 1:3:60 또는 1:3:400의 지질 비율 정화 MalFGK이 복합 ~ 2 nmol은 단백질에 혼합되어 있습니다. 최종 농도는 6 μM MalFGK 2, 18 μM의 MSP와 360 μM의 지질 (1:3:60) 또는 2.4 밀리미터 지질 (1:3:400)입니다. 최종 볼륨은 300 μl입니다. 최종 DDM 농도는 0.08 % (~ 1.6 ㎜)이다. 글리세롤의 마지막 농도가 지질의 양에 따라 달라집니다 추가 주변을 5~10% V / 대 유지 </lI&gt; <li> ~ 바이오 비드 정지의 50 μl는 튜브에 추가되고 혼합은 4 ° C.에 흔들 테이블에 하룻밤 incubated 수 있습니다 </li><li> 구슬이 중력에 의해 sedimented되며 솔루션은 가능한 한 많은 바이오 비즈를 피하기 위해 좁은 팁을 통해 pipetted 있습니다. </li><li> 침전물 대형은 20 분에 100,000 XG에 초 원심 분리에 의해 제거됩니다. 디스크는 TSG10 버퍼에 단계 2.4에서와 같이 겔 여과 크로마토 그래피에 의해 정화되어 있습니다. 디스크를 포함하는 분수는 풀링하고 나누어지는이 준비의 안정성을 테스트하기 위해 동일한 겔 여과 칼럼에 다시 주입해야합니다. </li><li> 정화 디스크는 오랜 기간 동안 -70 ° C에 저장 할 수 있습니다. 얼음에 해동 후, 디스크는 가능성이 침전물 제거하는 단계 5.5에서와 같이 초 원심 분리를 받게해야합니다. 나누어지는이 중요한 집계 나 강수량은 해동하는 동안 발생하지 않았 음을 보장하기 위해 겔 여과 크로마토 그래피에 의해 분석되어야한다과정입니다. </li></ol> 6. 기본 젤 전기 영동 <ol><li> nanodiscs 1 μM (~ 0.2 MG / ML)은 reconstitution (그림 3A)의 품질을 평가하는 기본 PAGE 6, 7 (트리스 - HCL 산도 8.8, 4-12 %)에 의해 분석됩니다. </li><li> 2 단지 MalFGK 남성과의 바인딩 (1 μM)가 nanodiscs에서 재구성은 기본 페이지 (그림 3A)에 의해 평가된다. </li><li> 전기 영동 후, 젤은 10 분에 Coomassie 파란색으로 물들어있는 것, 그리고 ~ 1 시간에 destained. </li></ol> 7. 동적 광 분산 (DL을) <ol><li> Nanodiscs는 0.1 ML / 분의 유량에서 Superdex 200 HR 300분의 10 열을 사용하여 TSG10 버퍼에 단계 2.4에서와 같이 겔 여과 크로마토 그래피에 의해 정화되어 있습니다. nanodiscs를 포함하는 분수는 풀링과 Amicon 원심 필터 ~ 10 MG / ML에 집중되어있다. </li><li> 샘플을 사용하여 DL을 분석하기 전에 (0.22 μm 필터) 두 번 필터링1 μl 내부 볼륨 석영 큐벳에서 DynaPro nanostar 악기 (와이어트 기술). 데이터는 직경과 입자의 분자량을 추정하기 위해 역학 소프트웨어 (와이어트 기술)를 사용하여이 마련되어 있습니다. </li></ol> 8. ATPase 측정 <ol><li> MalFGK 2 재구성 nanodiscs의 ATPase 활동이 colorimetric 분석 8을 사용하여 결정하고 있습니다. </li><li> 정화 디스크와 1 ㎜ ATP의 1 μM는 20 분에 대한 남성과 37 incubated ° C의 증가 금액과 함께 혼합되어 있습니다. 무기 인산염의 릴리스는 660 nm의에서 측정된다. </li><li> 출시 인산염의 금액은 인 표준 솔루션으로 생성 된 표준 곡선과 비교됩니다. </li></ol> 9. 대표 결과 nanodiscs는 겔 여과 크로마토 그래피 (그림 2A, 왼쪽)에 의해 정화되어 있습니다. 크로마토 그램은 보여줍니다 재구성 D의 대부분iscs (검은 색 추적) 하나의 피크로 elute, 반면에 과잉 지질 (빨간색 추적) 무효 볼륨의 elute로하고 폭 넓은 봉우리의 연속으로 만들어 디스크. nanodiscs의 품질은 더욱 네이티브 겔 전기 영동 및 동적 광 산란 분광법 (DL을)에 의해 분석됩니다. 지질의 과잉의 존재의 재구성 사람들은 비방 (그림 2A, 오른쪽)으로 마이그레이션 반면에 적절히 재구성 디스크는 젤에 날카로운 밴드로 마이그레이션됩니다. DL을하여 분석 디스크 인구가 11.4 nm의 (그림 2B)의 평균 직경이 균일 한 것을 보여줍니다. 재구성 디스크 DL을의 근사치에 따라 215 kDa의 명백한 분자량 있습니다. 초과 지질의 면전에서 재구성 샘플은 널리 비 균일 한 샘플에 대한 일반적인입니다, 100 nm의 주위 반경을 배포 표시됩니다. MalFGK이 단지의 품질은 ATPase의 기본 - 겔 전기 영동과 그 활동에 의해 평가된다측정 (그림 3). 말토오스 결합 단백질 남자는 MalFGK이 전송 9, 10에 높은 친 화성을 ​​바인딩합니다. 사용하지 않는 denaturing 겔 전기 영동,이 남성과 MalFGK 2 (그림 3A) 사이에 복잡한을 감지 할 수 있습니다. 수컷이 MalK이 ATPase 활동의 자극은 그림 3B에 표시됩니다. <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="그림 1"/> 1 그림. reconstitution 프로토콜에 대한 일반적인 흐름도. <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="그림 2"/> 그림 2. nanodisc 준비. A. 품질 관리 또는 높은 지질 비율 (1/3/400, 붉은 색 추적), nanodiscs의 젤 여과 분석 (Superdex 200 HR 3백분의 10 열)은 낮은 지질 비율 (검은 색 추적 1/3/60)에서 재구성. 동일한 디스크 준비 기본 겔 전기 영동. 몰kDa의 ecular 중량 표시가 표시됩니다. 동일한 디스크 준비 B. 동적 광 산란 분석. <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="그림 3"/> 그림 3. MalFGK 2 nanodisc 입자의 분석. A. 남성의 젤 이동 분석은 MalFGK 2 nanodisc 입자의 증가 금액과 incubated. MalFGK의 B. ATPase 활동 2 nanodisc 남성 농도의 함수로 입자.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein-induced+conversion+of+phosphatidylcholine+bilayer+vesicles+into+nanodisks.">Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks.</a> Biochim. Biophys. Acta (BBA). 1808, 606-613 (2011).</li><li>Nath, A., Trexler, A. J., Koo, P. K., Miranker, A. D., Atkins, W. M., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+spectroscopy+using+phospholipid+bilayer+Nanodiscs.">Single-molecule fluorescence spectroscopy using phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 472, 89-117 (2010).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+P450+in+Nanodiscs.">Cytochromes P450 in Nanodiscs.</a> Biochim. Biophys. Acta. 1814, 223-229 (2011).</li><li>Zhang, X. X., Chan, C. S., Bao, H., Fang, Y., Foster, L. J., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+and+SILAC-based+mass+spectrometry+to+identify+a+membrane+protein+interactome.">Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.</a> J. Proteome Res. , (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

우리는 nanodiscs에 말토오스 수송의 reconstitution에 대한 간단한 절차를 설명합니다. 전송은 ATPase 활성 상태이며 수용성 바인딩 파트너 남성과의 상호 작용은 (그림 3) 재현 할 수 있습니다. nanodiscs에 전송의 성공 reconstitution 추가 biophysical 및 생화학 분석 방법을 엽니 다. 특히 관심 MalK ATPase 및 세제의 말토오스 수송 활동, 리포좀과 nanodiscs 체계적인 분석됩니다. ABC의 이동 장치는 전송 사?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Amicon 울트라-4 50K 원심 필터 </td><td> Millipore </td><td> UFC805008 </td><td> 적절한 사용을 위해 제조업체의 규정을 따르 </td></tr><tr><td> 바이오 비즈 SM-2 흡착제 </td><td> 바이오 RAD </td><td> 152-3920 </td><td></td></tr><tr><td> 대장균 총 지질 </td><td> 벤티 폴라 지질 </td><td> 100500C </td><td> 클로로포름에 용해, 유기 용매에 따라 처리 </td></tr><tr><td> 니켈 세 파로스 HP 수지 </td><td> GE 헬스 케어 </td><td> 17-5268-01 </td><td></td></tr><tr><td> 인 표준 솔루션 </td><td>시그마 - 알드리치 </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20,061 </td><td></td></tr><tr><td> Superdex 200 HR 3백분의 10 </td><td> GE 헬스 케어 </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 표 I. 특정 시약. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 이름 </td><td> 구성 </td><td> 코멘트 </td></tr><tr><td> DDM 주식 </td><td> 10% w / V DDM </td><td> -20 ° C에서 milliQ 물과 저장소에 Resuspend </td></tr><tr><td> MalFGK이 주식 </td><td> 1-2 MG / ML 50 MM 트리스 - HCL, pH7.9 100 MM NaCl 10% 브이 / v 글리세롤 0.03 % w / V DDM </td><td> -70에서 저장 ° C 정화 후 </td></tr><tr><td> MSP 주식 </td><td> 10-15 MG / ML 50 MM 트리스 - HCL, pH7.9 100 MM NaCl 10% 브이 / v 글리세롤 </td><td> -70에서 저장 ° C &lt;1 ML의 aliquots에서 정화 이후 과도한 동결 / 해동 사이클을 방지 </td></tr><tr><td> 인지질 재고 </td><td> 5 NM E. 대장균은 지질를 집계 0.5 % w / V (10 ㎜) DDM 50 MM 트리스 - HCL, pH를 7.9 50 MM NaCl </td><td> 일주 4 ° C에서 저장 </td></tr><tr><td> TS 버퍼 </td><td> 50 MM 트리스 - HCL, pH를 7.9 50 MM NaCl </td><td> 4에 저장 ° C </td></tr><tr><td> TSG10 버퍼 </td><td> 50 MM 트리스 - HCL, pH7.9 100 MM NaCl 10% 브이 / v 글리세롤 </td><td> 4에 저장 ° C </td></tr><tr><td> TSG20 버퍼 </td><td> 50 MM 트리스 - HCL, pH8 100 MM NaCl 20% 브이 / v 글리세롤 </td><td> 4에 저장 ° C </td></tr><tr><td> TSGD 버퍼 </td><td> 50 MM 트리스 - HCL, pH7.9 100 MM NaCl 10% 브이 / v 글리세롤 0.03 % w / V DDM </td><td> 4 ° C에서 저장 불과 사용하기 전에 DDM을 추가 </td></tr><tr><td colspan="3"> 표 II. 솔루션 요리. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

nanodisc는 discoidal 입자 (~ 10-12 nm의 넓은)는 트랩 막 단백질 인지질 이중층의 작은 패치로.입니다 nanodisc 특히 리간드 - 수용체 상호 작용의 맥락에서, 막 단백질을 공부에 특히 매력적인 옵션입니다. 방법은 Sligar에 의해 개척 및 동료는 apolipoprotein A1에서 파생 설계 높은 - 나선형 비계 단백질의 amphipathic 특성에 기반을두고 있습니다. 극지방의 수성 환경에 직면 반면 비계 단백질의 소수성 얼굴은 지질 이중층의 지방산 아실 사이드 체인과 상호 작용할 수 있습니다. 입자가 작은 균질하고 수용성이기 때문에 nanodiscs의 막 단백질의 분석은 리포좀에 비해 상당한 장점이 있습니다. 또한, 일반적으로 수용성 단백질에 예약 생​​화학 및 biophysical 방법이 적용하고 막 양쪽에서 할 수 있습니다. 이 시각 프로토콜에서, 우리는 잘 charac의 단계별로 reconstitution을 제시terized 세균 ABC의 전송, 남성 MalFGK이 복잡. 디스크의 형성은 세제의 진보적 인 제거에있어 소수성 상호 작용에 따라 자기 조립 과정입니다. 우리는 필수적인 단계를 설명하고 우리는 집계 및 큰 polydisperse 리포좀과 같은 입자의 형성을 제한하기 위해 올바른 단백질 - 투 - 지질의 비율을 선택의 중요성을 강조 표시합니다. 간단한 품질은 디스크가 올바르게 재구성되었는지 확인 겔 여과 크로마토 그래피, 기본 겔 전기 영동 및 동적 광 산란 분광법으로 제어합니다.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs는 인지질 이중층의 작은 패치로 막 단백질을 포함 작은 원반 모양의 입자입니다. 우리는 디스크에 MalFGK2 수송의 단계별 설립을 보여줍니다 시각적 프로토콜을 제공합니다.

Nanodisc 지질 입자로 ABC의 전송의 Reconstitution을위한 단계별 방법

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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Research

JoVE Journal

Biology

25.3K 조회수.  University of British Columbia. Nanodiscs는 인지질 이중층의 작은 패치로 막 단백질을 포함 작은 원반 모양의 입자입니다. 우리는 디스크에 MalFGK2 수송의 단계별 설립을 보여줍니다 시각적 프로토콜을 제공합니다.

비디오: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

표현, 세제 Solubilization 및 멤브레인 전송, MexB의 Multidrug 저항 단백질의 정제

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

연구

생물학

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

구조 및 기능 연구를위한 지질 막에 Kv 값 채널의 재구성

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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 ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

절차 식물 추출물에서 분산 된 입자의 제거를위한 응집제의 효율성을 평가하는

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

효율적인 체 외에서 이항 메서드인 Transcriptionally 규제 자 아름다움 시스템 통합 결함이 Lentiviral 벡터에 포장 하 여

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-지우기: 조직 지방 조직의 3 차원 이미징 방법 삭제

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

독성 테스트를 위해 공기 중 입자에 대한 사실적이고 반복적인 흡입 노출을 위한 공기 액체 인터페이스 기관지 상피 모델

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...