Name: one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)
Uploaded: 2014-04-20T14:00:00.000+00:00
Duration: 18 min 27 s
Description: Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

우리는 기꺼이 시니 Miettinen, 미나 Pöllänen 및 한인 Rautavesi의 기술 지원을 인정합니다. 우리는 HEK에게 녹음 장비를 제공하는 트윈 연쇄상 구균 - 태그 GFP 및 페카 Evijärvi을 표현하는 293 세포를 제공 헬카 Nurkkala 감사합니다. 이 작품은 핀란드의 아카데미에 의해 투자되었다, 번호 138329, 134684 및 258978을 부여합니다.

1. 비스와 패 혈성-Tactin 폴리 메타 크릴 수지 (술포 숙신 이미 딜)의 가교 수베 (BS3) <ol><li> 실온 BS3 가교제의 2 ㎎을 함유 한 밀봉 된 마이크로 튜브를 평형화. 주의 : BS3는 유해 물질이다. 보호 장갑과 고글을 착용 할 것. </li><li> 재현 탁 패 혈성-Tactin의 폴리 메타 크릴 레이트 수지 (100 MM 트리스 - 염산, 산도 8.0의 50 % 현탁액, 1 mM의 EDTA, 150 mM의 염화나트륨)에 의한 간단한 격렬한 흔들림 즉시에 피펫 팁을 사용하여 스핀 컬럼으로 서스펜션의 600 μl를 전송 끝은 잘라. </li><li> 실온에서 30 초 동안 1,500 XG에 원심 분리기. 흐름을 통해 폐기하고 열을 인산염 완충 생리 식염수 (PBS)의 450 μl를 추가합니다. </li><li> 완전히 PBS로 트리스 버퍼를 교체하고 마지막으로 원심 분리 한 후 pH를 8.0로 조정 PBS 430 μL에 수지를 떠나 이전 단계 2를 번 더 반복합니다. </li><li> (100)를 포함하는 피펫 팁 BS3으로 모세관의 호일에 구멍을1, 초순수의 L. 부드럽게 아래로 피펫에 의해 물에 BS3 분말을 녹여 바로 스핀 열 용액 20 μl를 추가합니다. 가교 반응에 BS3의 최종 농도는 ~ 1.2 ㎜이다. </li><li> 실온에서 30 분 동안 열을 돌립니다. 수지가 BS3 솔루션을 적절하게 혼합되어 있는지 확인합니다. </li><li> 반응을 해소하기 위해, 3M 트리스 - 염산, 산도 7.5의 10 μl를 추가하고 실온에서 또 다른 15 분 동안 열을 돌립니다. </li><li> 실온에서 30 초 동안 1,500 XG에 원심 분리기. 흐름을 통해 폐기 및 트리스 450 μL의 가교 수지를 재현 탁은 트윈 20 (TBST)와 완충 식염수. 원심 분리를 반복하고 단계를 두 번 더 씻는다. 마지막 단계에서, TBS 450 μL에 재현 탁 수지. </li><li> 끝으로 피펫 팁을 사용하여 새로운 튜브에 수지 현탁액을 전송 차단. TBS의 또 다른 450 μL와 컬럼에 남아있는 수지를 재현 탁하고 같은 튜브에 전송할 수 있습니다. 반복 일한 번 더 튜브에 열에서 수지의 최대 전송을 보장하기 위해 전자 마지막 단계. </li><li> 관을 실온에서 10 분간 방치하고 과량의 TBS를 제거하여 600 μL로 볼륨을 조절하자. 수지 (권장) 즉시 사용할 준비가 또는 냉동하지 않고, 4 ° C에 저장할 수 있습니다. </li></ol> 2. 교차 연결된 연쇄상-Tactin 폴리 메타 크릴 레이트 수지에 트윈 연쇄상 구균 - 태그 미끼 단백질 및 관련 단지의 바인딩 <ol><li> 4 ° C에서 10 분 동안 17,000 XG에서 버퍼를 결합하고 새로운 튜브에 뜨는을 전송의 미끼 단백질 시료의 원심 분리기 1 ML. </li><li> , 패 혈성-Tactin 수지에 내인성 바이오틴 단백질의 결합을 최소화 100 μg / ML의 최종 농도에 아비딘을 추가하고 4 ℃에서 15 분 동안 회전하려면 <ol><li> 단계 1.10에서 가교 수지는 4 ° C에서 장기간 보관 한 경우에, FO 400 XG에서 원심 분리에 의해 수지를 상환4 ° C에서 R 1 분, 상층 액을 버리고 TBST 1 ㎖​​로 수지를 세척한다. 원심 분리를 반복 세척 먼저 TBST로 다음 TBS와 두 번 더, 단계를 반복합니다. 4 ° C에서 1 분 400 XG에 튜브를 원심 분리기 및 초과 TBS를 제거하여 원래 볼륨을 조정합니다. </li></ol></li><li> 볼 텍싱에 의해 가교 된 패 혈성-Tactin 수지에 resuspend. 즉시 컷 피펫 팁을 사용하여 미끼 단백질 샘플이 들어있는 튜브에 수지 현탁액의 50 μl를 추가하고 4 ℃에서 또 다른 30 ​​분 동안 회전 </li><li> 기다리는 동안, 55 ° C에 thermomixer을 설정하고 단계 3.1에서 사용하기 위해 용출 버퍼 500 μl를 예열. </li><li> 4 ℃에서 1 분 400 XG에 원심 분리기 뜨는을 취소하고 미리 냉각 세척 버퍼 1 중 1 ㎖를 4 ° C에서 5 분 동안 회전에 수지를 씻어. 원심 분리를 반복하고 단계를 세 번 씻는다. 마지막 단계에서, 세척 버퍼 # 2 250 μL의 수지를 재현 탁. </li><li> TR<html> 신선한 스핀 열 수지 현탁액을 ansfer. 세척 버퍼 # 2의 또 다른 250 μL의 튜브에 남아있는 수지를 재현 탁하고 같은 C 럼에 전송할 수 있습니다. </li><li> 4 ° C에서 3 분 400 XG에 원심 분리기, 흐름을 통해 폐기하고 신선한 돌고래 코 2 ㎖ 튜브에 열을 전송합니다. 아래의 용출 단계로 바로 진행합니다. </li></ol> 특정 단백질 복합체의 3. 용출 <ol><li> 스핀 컬럼에 2.4 단계에서 예열 된 용출 버퍼 150 μl를 추가합니다. </li><li> 1,400 rpm으로 흔들어, 55 ° C에서 5 분 thermomixer에 품어. </li><li> 실온에서 1 분 1,500 XG에 원심 분리기. </li><li> 열을 버리고 ≤ -20 ℃에서 정제 된 표적 단백질을 저장 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="598"><tbody><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 인산염 완충 생리 식염수 (PBS) </td></tr>0 &quot;&gt; <td height="20" style="height:20px;width:299px;"> 나 2 HPO 4 </td><td style="width:299px;"> 10 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> KH 2 PO 4 </td><td style="width:299px;"> 2mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 137 밀리 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 의 KCl </td><td style="width:299px;"> 2.7 mM의 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> (섹션 1.4의 pH 8.0) 달리 명시되지 않는 pH는 7.4로 조정 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 트리스 (TBS)를 완충 식염수 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH 7.4의 </td><td style="width:299px;"> 50 mM의 </td></tr><tr height="20"><td hei<html> GHT = &quot;20&quot;스타일 = &quot;높이 : 20px의 폭 : 299px;&quot;&gt; 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 트리스는 트윈 20 (TBST)와 완충 식염수 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH 7.4의 </td><td style="width:299px;"> 50 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트윈 20 </td><td style="width:299px;"> 0.1 % (V / V) </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 바인딩 버퍼 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td>X; &quot;&gt; 550 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 불화 나트륨 </td><td style="width:299px;"> 5 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.5 ㎜ </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 글리세린 </td><td style="width:299px;"> 10 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1 ㎜ </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 버퍼 # 1을 씻으 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 500 밀리미터 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 불화 나트륨 </<html> TD&gt; <td style="width:299px;"> 5 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> EDTA </td><td style="width:299px;"> 0.4 ㎜ </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 이게 팔 CA-630 </td><td style="width:299px;"> 0.2 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 글리세린 </td><td style="width:299px;"> 5 % (V / V) </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> PMSF </td><td style="width:299px;"> 0.1 ㎜ </td></tr><tr height="20"><td colspan="2" height="20" style="height:20px;width:597px;"> 버퍼 # 2를 세척 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 염화나트륨 </td><td style="width:299px;"> 150 밀리미터 </td></tr><tr height="20">2 &quot;높이 =&quot;20 &quot;스타일 =&quot;높이 : 20px의 폭 : 597px; &quot;&gt; 용출 버퍼 </td></tr><tr height="20"><td height="20" style="height:20px;width:299px;"> 트리스 - 염산, pH를 8.0 </td><td style="width:299px;"> 25 mM의 </td></tr><tr height="22"><td height="22" style="height:22px;width:299px;"> SDS </td><td style="width:299px;"> 1 % (W / V) </td></tr></tbody></table> 표 1. 본 연구에 사용 된 버퍼.

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상기 프로토콜은 비오틴 또는 강한 변성제를 포함하지 않는 임의의 적절한 버퍼에 대한 관심 및 관련 착체의 트윈-Strep의 태그 미끼 단백질을 정제하는데 사용될 수있다. 프로토콜의 현재 버전에서, 결합 및 세척이 높은 염과 비 이온 세제의 존재하에 비교적 엄격한 조건하에 수행된다. 이 적은 배경을 초래하지만, 깨지기 쉬운 단백질 복합체는 이러한 조건에서 해리 수 있습니다. 이러한 낮은 친?...

최근 몇 년 동안, 패 혈성 태그 기술은 단백질 체학 및 구조 생물학을 포함한 생물 의학 연구의 많은 분야에서 널리 사용되고있다. 짧은 Strep의 태그 펩티드로 재조합 단백질의 융합에 의존이 단백질 정제 기술은, 패 혈성-Tactin 향상된 펩티드 결합능과 스트렙 타비 딘의 유전 공학적 변형을 운반 궁합 행렬들의 출현으로 성숙. 1,2 트윈 패 혈성 태그 또는 SIII 태그, 전시 재조합 단백질을보다 효율적으로 정화를 보장, 단 하나의 패 혈성 태그를 포함하는보다 연쇄상 구균 - Tactin 행렬에 대한 높은 선호도와 지정 패 혈성 태그 II의 두 복사본을 포함하는 융합 단백질 연관된 바인딩 파트너. 그러나, 패 혈성-Tactin에 트윈 연쇄상 구균 - 태그 단백질의 높은 친화력은 또한 단점이있다. 과량의 비타민 B 복합체와 단백질의 경쟁 용출 대상 단백질 수율 감소로 이어지는, 불완전 할 수 있습니다. M광석 효율적인 대안 SDS와 용출이지만, 프로테오믹스 분석 분석 호환되지 수지에서 발표 연쇄상-Tactin와 바람직하지 않은 샘플의 오염으로 이어집니다. 이 논문은 제 화학적 가교에 의하여 패 혈성-Tactin의 수지 - 결합 사량을 안정화 한 후 얻어진 가교 수지와 트윈 연쇄상-태그 된 단백질과 관련 복합체를 용출 SDS를 사용하여이 제한을 극복하는 기술을 제시한다. 따라서, 충분한 단백질 수율함으로써 질량 분석에 의한 추가 분석을 허용 연쇄상-Tactin 샘플 오염없이 달성 될 수있다. 이 방법은 표면에 노출 된 SIII 태그 3 트윈 패 혈성 태그 (아미노산 서열 WSHPQFEK (GGGS) 각각 3 WSHPQFEK 및 SAWSHPQFEK (GGGS) 2 GGSAWSHPQFEK) 어떤 재조합 융합 단백질의 정제를 위해 적당하다. 단백질은 동물, 식물 또는 세균 기원 일 수 있으며 전체 셀 중 하나로부터 단리 될 수있다해물이나 농축 세포 기관의 분수. 예를 들어, 우리는 여기에 PVA에 감염된는 담배 benthamiana 식물의 핵 부분에서 감자 바이러스 A (PVA) 4 SIII 태그가 단백질 VPG-PRO의 정화에 대해 설명합니다. 이전에 다음과 같이 수정하여, 5 바와 같이 핵 분획을 단리 하였다 : 세포를 포름 알데히드로 처리되지 않은, 나트륨 부티레이트를 5 mM의 불소화 나트륨으로 모든 버퍼에 치환하여, 완전 프로테아제 억제제 PMSF로 치환하고, 추출 트리톤 X-100 농도 버퍼 # 2는 0.3 %로 감소 하였다 (V / V) 및 (추출 버퍼 # 3) 자당 쿠션을 통해 원심 분리하여 얻은 핵 펠릿 사전 냉장 바인딩 버퍼의 1.45 ㎖에 재현 탁하고, 4 ℃에서 1.5 시간 동안 회전 SIII-태그 미끼 단백질과 관련된 단지 (미끼 단백질 샘플)를 포함하는 결과 핵 추출물 (섹션 2 참조) 아래에 설명 된 프로토콜에 따라 처리되었습니다.

<table><tbody><tr><td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2 mg</td><td>Pierce/Thermo Scientific</td><td>21585</td><td>&lt;a href=&quot;http://www.fishersci.com&quot;&gt;www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.&lt;/a&gt;</td></tr><tr><td>Strep-Tactin MacroPrep resin (50% suspension)</td><td>IBA</td><td>2-1505</td><td>&lt;a href=&quot;http://www.iba-lifesciences.com/&quot;&gt;www.iba-lifesciences.com&lt;/a&gt;</td></tr><tr><td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &amp;mu;m, non-sterile</td><td>Costar (Corning)</td><td>8163</td><td>&lt;a href=&quot;http://www.corning.com/lifesciences/&quot;&gt;www.corning.com/lifesciences/&lt;/a&gt;</td></tr><tr><td>Dolphin-nose tubes</td><td>Costar (Corning)</td><td>3213</td><td>&lt;a href=&quot;http://www.corning.com/lifesciences/&quot;&gt;www.corning.com/lifesciences/&lt;/a&gt;</td></tr><tr><td>Avidin&amp;nbsp;</td><td>IBA</td><td>2-0204</td><td>&lt;a href=&quot;http://www.iba-lifesciences.com/&quot;&gt;www.iba-lifesciences.com&lt;/a&gt;</td></tr></tbody></table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

설계 스트렙 타비 딘 (연쇄상-Tactin)를 운반하는 수지에 패 혈성 태그가 융합 단백질의 친 화성 정제는 생리 학적 조건에서 단백질 복합체의 분리를위한 널리 사용되는 방법이되고있다. 트윈 패 혈성 태그 또는 SIII 태그를 지정 패 혈성 태그 II의 두 복사본을 포함하는 융합 단백질 따라서보다 효율적인 단백질 정제를 허용, 단 하나의 패 혈성 태그를 포함하는 사람에 비해 연쇄상-Tactin에 대한 높은 친 화성의 장점이 있습니다. 그러나, 이러한 장점은 비오틴과 트윈 연쇄상 구균 - 태그 단백질의 용출이 낮은 단백질 복구에 이르는, 불완전 할 수 있다는 사실에 의해 상쇄된다. 복구 극적 도데 실 황산나트륨 (SDS)으로 변성 용출을 사용함으로써 개선 될 수 있지만 하류 단백질체 분석으로 분석 호환되지 수지로부터 방출 연쇄상-Tactin 오염을 샘플링 리드. 이 한계를 극복하기 위해, 우리는 방법 연쇄상-Tactin의 그것에 수지 결합 사량을 개발했습니다제 공유 비스 (술포 숙신 이미 딜) 수베 레이트 (BS3)와 가교하고, 생성 된 가교 된 수지를 다음 번의 배치 정제 단계에서 목적 단백질 복합체를 정제하는 데 사용함으로써 안정화된다. 패 혈성-Tactin 오염의 부재는 질량 분석에 의한 하류 단백질 분석을 허용하는 동안 SDS와 효율적인 용출, 좋은 단백질 복구를 보장합니다. 개념의 증거로서, 우리는 바이러스에 감염된 N.의 핵에서 VPG-프로 SIII 태그가 바이러스 성 단백질의 정제를 위해 여기 프로토콜을 설명 BS3으로 가교 패 혈성-Tactin의 폴리 메타 크릴 레이트 수지를 사용한 benthamiana 식물. 동일한 프로토콜은 또한 어떤 트윈 연쇄상-태그 단백질을 정제 및 생리 바인딩 파트너를 특성화하는데 사용될 수있다.

정화 절차를 개략적으로 서로 다른 기존의 정제 방법과 관련된 문제의 표현과 함께,도 1에 도시되어있다. <img alt="그림 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 그림 1. 정화 절차의 도식 표현. 워크 플로는 공유가 가교 수지에 트윈 연쇄상 구균 - 태그 미끼 단백질과 연관된 복합체의 결합...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

방법은 (연쇄상 구균 - Tactin) 수지가 공유 비스 (술포 숙신 이미 딜) 수베 (BS3)와 가교 수정 스트렙 타비 딘에 트윈 패 혈성 태그가 융합 단백질 및 특정 단지의 효율적인 정제에 설명되어 있습니다. 방법은 빠른 속도, 좋은 표적 단백질의 회수 및 고순도의 이점을 갖고, 질량 분석법에 의한 후속 분석과 호환된다.

한 단계 연쇄상-Tactin 수지 비스 (술포 숙신 이미 딜)로 가교 수베 (BS3)에 트윈 연쇄상 구균 - 태그 단백질과 그들의 단지의 정화

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

18.7K 조회수.  University of Helsinki.  방법은 (연쇄상 구균 - Tactin) 수지가 공유 비스 (술포 숙신 이미 딜) 수베 (BS3)와 가교 수정 스트렙 타비 딘에 트윈 패 혈성 태그가 융합 단백질 및 특정 단지의 효율적인 정제에 설명되어 있습니다. 방법은 빠른 속도, 좋은 표적 단백질의 회수 및 고순도의 이점을 갖고, 질량 분석법에 의한 후속 분석과 호환된다. 

비디오: One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

진핵 세포에서 단백질 복합체의 탠덤 선호도 정화

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

연구

생화학

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

감염된 세포의 염색질에서 인플루엔자 바이러스 Ribonucleoprotein 컴플렉스의 동질 정제

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

면역학 및 감염병학

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

생물학

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

무세포 재구성을 위한 재조합 셉틴 복합체의 정제 및 품질 관리

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

작은 Bispecific 선호도 태그에 의해 용이 직교 단백질 정제

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

탠덤 동질 정제를 사용하여 단백질 나누기 파트너의 식별

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

연쇄상 구균 뮤탄에서 높은 분자 질량 단백질의 정화

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST - 그의 정화 : 전체 길이 정제 된 단백질을 항복 2 단계 친화력 정화 프로토콜

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

탠덤 선호도 태그 방법을 사용하여 구조 연구에 대한 기본 공단의 정화

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...