이 프로젝트는 Knut와 앨리스 발렌 버그 재단과 스웨덴 연구위원회에 의해 투자되었다.

1. 대상 gene/ABDz1-tagged 융합의 복제가 구축 <ol><li> 플라스미드 표현의 대상 유전자 (플라스미드 포함 ABDz1이 물질 전송 계약을 통해 자유롭게 사용할 수 있습니다)의 N - 터미널 내고 적합한 제한 사이트에 둘러싸인 ABDz1 유전자의 PCR 단편을 준비합니다. </li><li> 다니엘 정화 표현 벡터 및 적절한 반응 버퍼에서 선택한 제한 효소로 PCR 파편. 결합하기 전에 제품을 정화. </li><li> 관심의 유전자를 포함하는 발현 벡터에 제한 ABDz1 조각 Ligate와 E로 내고 제품을 변환 대장균 (원래 메서드에 RR1ΔM15 변형이 8 사용되었습니다). 선택에 적합한 항생제로 보충 한천 플레이트에 변형 세포를 확산. </li><li> PCR 화면 몇 식민지와 순서는 DNA 배열에 의해 결과 표현 카세트를 확인합니다. </li><li> 순서 verif의 야간 문화 플라스미드 준비처음엔 식민지와 선호하는 표현 스트레인 (인간 유전자에 의해 암호화된 단백질의 생산을 향상시키기 위해 플라스미드 pRARE를 호스팅하는 로제 원래 방법 E. 대장균 (DE3)에 사용되었다)로 변환. </li></ol> 2. 단백질 표현 <ol><li> 5g / L 효모 추출물과 적절한 항생제로 보충 tryptic 간장 국물의 10 ML에 하나의 박테리아 식민지의 예방. ° C 야간 37 150 rpm으로 품어. </li><li> 100 ML 신선한 매체 숙박 문화의 한 ML을 예방하고 세포 로그 성장 단계에 도달했을 때 단백질 표현을 (LAC 오스 오 페론이 활용되었을 때 이소 프로필 - β - D - thiogalactoside 원래 1 ㎜) 유도. </li><li> 원심 분리에 의해 ° C 야간 및 수확 세포 25 150 rpm으로 품어. </li></ol> 3. 직교 친화 정화 <ol><li> 25 ML 실행 버퍼 (25 MM 트리스 - HCL, 200 MM NaCl, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 0.05 % (W / V) 십대 초반 20, 산도 8.0)에 펠렛을 Resuspend하고 일을 방해60 % 진폭 3 분 1.0/1.0 펄스에서 sonication하여 전자 세포. 샘플을 원심 더욱 정화하기 전에 뜨는을 (0.45 μm의)이있는 대상 단백질을 필터링합니다. </li><li> 적당한 단백질 정제 시스템 1 ML / 분 버퍼를 실행하는 10 열 볼륨 (CV)와 이전에 공급자의 권고에 따라 HSA와 결합 중 1 ML HSA 세파 로스 칼럼이나 보건국 활성화 열을, 평형. 0.5 ML / 분 세균성 lysate를로드하고 다음 유량 1 ML / 분 재설정하십시오. </li><li> 세척 버퍼 5 CV (5 MM NH 4 AC, 산도 5.5) 다음에 실행하는 버퍼의 5 CV와 컬럼을 씻으십시오. </li><li> 1 ML / 분 용출 버퍼 (0.5 M HAC, 산도 2.5)와 함께 샘플을 Elute 더욱 정화를위한 eluted 정상에서 분수를 선택하는 280 nm의에서 모니터 흡수를 분수를 수집합니다. </li><li> 최고의 단백질 농도와 풀 분수는 확실히 실행 버퍼에 두 번 희석 (20 MM 인산, 150 MM NaCl, 산도 7.2)와산도 주변 중립 있는지 확인하십시오. 산도 필요한 경우를 높이기 위해 1 M 트리스 - HCL pH를 8을 추가합니다. </li><li> 1 ML / 분 물론 실행 버퍼의 10 이력서와 equilibrated되었습니다 1 ML의 HiTrap의 MabSelect 물론 열을 0.5 ML / 분 샘플을로드합니다. 1 ML / 로딩 후 분 흐름 속도를 재설정합니다. </li><li> 물론 실행 버퍼 5 CV (5 단계)로 컬럼을 씻고 0.2 M HAC, 산도 2.7과 단백질을 elute. 민감한 대상 단백질에 대한 eluate는 트리스 - HCL를 추가하여 수령시 직접 무력화 수 있습니다. </li></ol> 색층 단계 MabSelect에서 eluate을 반대하는 경우에는 물론 열이 버퍼 (3.1 단계)을 실행에 희석하고 산도가 1 M 트리스 - HCL의 추가에 의해 주위에 8 증가 수 있습니다. 물론 매트릭스가 두 번째 단계에서 고용되었을 때 일반적으로 좁아 봉우리가 관찰됩니다. 4. 나트륨 dodecyl 황산염의 polyacrylamide 겔 전기 영동 (SDS - PAGE)에 의한 순도 평가 <ol><li> 에서 정화 분수로드원하는 경우, 질량 분석법에 의한 정화 제품의 분자량을 분석한 다음, SDS - PAGE을 감소합니다. 그것은 ABDz1에서 무료 시스테인이 아닌 감소 조건에서 제품의 dimerization을 일으킬 수 있으므로 충분히 샘플을 줄이는 것이 중요합니다. </li></ol> 5. 대표 결과 : 원리의 증거로서, ABDz1 태그에 의해 용이 직교 친화 정화는 서로 다른 용해도 클래스, 분자 무게 및 isoelectric 포인트 (표 1)을 나타내는 세 인간의 목표 단백질에 대한 평가되었다. ABDz1 유전자가 유전자 대상 유전자에 융합되었고 구조가 E. 대장균으로 표현했고 그림 2는 연속적 방법의 모든 단계에 대한 플로우 차트를 보여줍니다. 직교 프로토콜에 의해 정화에 따라 다른 지점에서 수집된 샘플을 SDS - PAGE (그림 3)에 의해 분석되었다. 결과는 명확하게 듀얼 태그와 두 매우 구체적인 정화 단계의 유틸리티를 보여줍니다. 합리적인 순결은 AC하더라도로 젤에서 본 최초의 정화 단계 이후 hieved, 연속 단계는 물론 매우 까다로운 애플 리케이션에 적합한 매우 순수한 제품을 산출. <img alt="그림 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> 그림 1. bispecific ABDz1 태그의 선택에 사용되는 조합 도서관의 디자인. 46 아미노산 알부민 결합 도메인 안정적인 세 나선 번들로 폴드 그리고 그것은 주로 두 번째 나선형에있는 인간 혈청 알부민에 대한 구속력이 사이트가 포함되어 있습니다. 유전자 첫 번째와 세번째에 위치하고 나선 열한 표면에 노출된 아미노산을 randomizing함으로써, 소설 바인딩 표면은 설계되었습니다. 열한 위치는 그림에 표시된 및 Kraulis 외에 따라 번호가 9.되었습니다. 파지에 대한 표현 조합 도서관은, bispecific ABDz1 분자가 확인되었다 포도상 구균 단백질의 Z - 도메인의 이합체에 대한 biopanning 따라. <iMG 고도 = &quot;그림 2&quot;SRC = &quot;/ files/ftp_upload/3370/3370fig2.jpg&quot;/&gt; 그림 2. 직교 친화 정화 프로토콜에 대한 단순 플로우 차트. ABDz1 시퀀스를 포함하는 PCR - 단편은 적절한 발현 벡터에 대한 관심의 유전자와 (N - 말기) 출혈도 잡았 있습니다. 내고 제품은 E.으로 변환됩니다 순서 확인 및 플라스미드 준비 대장균. 표현 호스트로 변환 후, 재조합 단백질 표현을위한 대규모 문화는 초기 야간 문화에서 설정됩니다. 박테리아는 원심 분리에 의해 수확 및 대상 단백질을 포함하는 박테리아 lysate를 만들어 sonication하여 lysed 있습니다. 잔류 고체 입자를 제거하는 여과 단계 후, lysate는 연속 두 정화 단계를 진행하여 액체 처리 시스템에 직교 친화 정화를 받게됩니다. 모두 정화 단계에서 Eluted 봉우리는 샘플과 정결을 평가하기 위해 SDS - PAGE에 의해 평가하고 t에 비해 아르그는 정화하기 전에 lysate. <img alt="그림 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> 그림 3. 표명과 ABDz1과 융합의 정화 대상 단백질의 SDS - PAGE 분석. 다른 속성을 나타내는 ABDz1, 그리고 ABDz1 태그 자체 융합으로 표현 세 단백질의 세균성 lysate에서 가져온 샘플은 물론 열 MabSelect 다음 HSA - 열에 정화에서 해당 봉우리에서 샘플과 함께 수집된 (A) . 레인 1-4 9-12 MabSelect 물론 정화 (두 번째 단계)에서 정화, HSA - 정화의 차선 5-8 (첫 단계)와 골목길 전에 lysates에서 샘플을 나타냅니다. ABDz1 - 141377, ABDz1 - HT875, ABDz1 - HT2375과 ABDz1 : 샘플은 다음과 같은 순서로로드됩니다. 또한, 동일한 컬럼에 대한 역순으로 정화 같은 lysates로부터 얻은 샘플을 분석했다 (B). 레인은 정화, 차선 4-6 F 전에 lysates에서 샘플을 1-3 대표롬 MabSelect 진짜로 정화 (첫 단계)와 HSA - 정화 (두 번째 단계)에서 레인 7-9. 샘플은 (A)하지만 태그 자체가 포함되지 않은에서와 같은 순서로로드했습니다. 그 결과는이 두 단계 방법에 관계없이 단계가 적용되는 순서의 고도 정화 단백질을 산출 것이 분명하다. <table border="1"><tbody><tr><td> 이름 B </td><td> 대상 단백질 Uniprot C </td><td> 분자 중량 (kDa) </td><td> 용해도 클래스 D </td><td> 분자량 융합 제품의 (kDa) </td><td> Isoelectric 지점 융합 제품의 </td></tr><tr><td> 141,377 </td><td> B7Z315 </td><td> 17.4 </td><td> 4 </td><td> 23.7 </td><td> 8.5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10.8 </td><td> 3 </td><td> 17.1 </td><td> 4.9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8.9 </td><td> 5 </td><td> 15.2 </td><td> 6.8 </td></tr></tbody></table><ol class="lalpha"><li> 혼자 ABDz1 태그의 분자량 6.3 kDa, isoelectric 포인트 6.7입니다. </li><li> 대상 단백질은 Uniprot 단백질의 일부​​를 나타냅니다. </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> . </li><li> N - 터미널과 단백질 조각 그의 6 ABP 10 용해도 클래스. </li></ol> 표 1. ABDz1 태그와 대상 단백질과 융합 제품은 원칙적으로 연구의 증거로 평가. 다양한 분자량, 용해도 및 isoelectric 포인트 세 가지 독특한 인간의 단백질은 직교 친화 방식에 의해 표현과 정화를 위해 선택되었습니다.

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관심 없음 충돌 선언하지 않습니다.

여기에 제시 직교 친화 정화 프로토콜은 표적 단백질의 광범위한 효율적인 정화 수 있습니다. 소설 바인딩 표면과 알부민 결합 도메인의 고유 바인딩 사이트를 결합하여, 작은 bispecific 단백질 태그가 개발되었다. 그것이 단순히 어떤 선호하​​는 발현 벡터에 대한 관심의 단백질로 복제 및 융합 표현 할 수 있기 때문에 태그를 활용하는 것은 매우 간단합니다. 대부분의 실험실에서 사용할 수있는 표준 장비는 두 단계 단백질 정화에 대한 고용 수 있습니다. ABDz1 태그의 기능을 효율적으로 두개의 바인딩 표면을 노출하기 위해 도메인의 올바른 접힘에 의존 때문에, 방법은 용해 형태로 표현 단백질로 제한하고 denaturing 조건 하에서 purifications 적합하지 않습니다. 그들은 MabSelect 물론 매트릭스에서 Z - 도메인에 ABDz1의 바인딩을 방해 이후 감소 요원도 피해야한다. 직교 친화 purifica기는 매우 여러 까다로운 응용 프로그램에서 단백질을 정제의 요구 사항을 충족하기 위해 전통적인 방법에 대한 유용한 대안을 제공합니다. 동시에,이 방법은 다른 정화 전략이 연속적으로 사용되는 조합 태그에 대한 필요가 없습니다. 기본 목표 단백질을 필요로하는 어플 리케이션의 경우, 프로 테아제의 절단 사이트는 가능한 ABDz1 태그 11 효소 제거를 렌더링 도입 수 있습니다. 또한 ABDz1 태그는 날짜 설명한 최초의 bispecific 중소 접힌 단백질 도메인이다. 그 두 대상 특정 친화 상호 작용에 따라 정화 전략을 제공하는 지 유일하다. 앞으로 그것은 쉽게 사용할 수 있으며 저렴한 색층 수지와 호환되는 새로운 바인딩 표면을 가지고 소설 bispecific 태그를 개발하여이 개념을 확장하는 흥미로운 것입니다. 예를 들어, 태그가 호환 기본 잔류물와 알부민 바인딩에 관련된 아미노산을 대체하여교환 크로마 토그래피에 도달 수 있습니다. 이와 유사한 개념은 각각 Z 산 12 음이온과 양이온 교환과 호환 Z 기본 13으로 알려진 태그를 생성하기 위해 Z - 도메인을 담당 엔지니어링에 의해 효과가 입증되었습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> E. 대장균 RR1ΔM15 </td><td> 미국 유형 문화 수집 </td><td> 35,102 </td></tr><tr><td> 로제 E. 대장균 (DE3) </td><td> Novagen </td><td> 70,954 </td></tr><tr><td> Tryptic 콩 국물 </td><td> Difco </td><td> 211,822 </td></tr><tr><td> 효모 추출물 </td><td> Difco </td><td> 212,720 </td></tr><tr><td> Vibra 셀 sonicator </td><td> Sonics 및 자료 </td><td> - </td></tr><tr><td> HSA 세파 로스 </td><td> Pharmacia 생명 공학 </td><td> 전 제품 </td></tr><tr><td> 물론 HiTrap MabSelect </td><td> GE 헬스케어 </td><td> 11-0034-93 </td></tr><tr><td> HiTrap 보건국 - 활성화 HP 칼럼 </td><td> GE 헬스케어 </td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

프로토콜은 합리해야 재조합 단백질의 정화와 관련된 높은 비용으로 인해. 높은 처리량 노력 타겟 단백질 특정 최적화 1 필요하지 않은 일반적인 방법에 대한 수요가있다. 이것을 달성하기 위해, 유전자의 관심 유전자에 융합 될 수있는 정화 태그는 일반적으로 2 사용됩니다. 가장 널리 사용되는 화성 처리 기본 및 denaturing 조건 하에서 3 모두 정화에 적합합니다 hexa - 히스티딘 태그입니다. 태그를 생산을위한 대사 부담이 낮은이지만, 경쟁 친화 크로마 토그래피 기반 전략 1,2 높은 특이성으로 제공하지 않습니다. 여기, 46 아미노산에 두 개의 서로 다른 바인딩 사이트 bispecific 정화 태그는, 작은 단백질 도메인이 개발되었습니다. 알부민 결합 도메인 구균 단백질 G에서 파생된 인간 혈청 알부민에 강한 고유의 친화력을 가지고있다(HSA). 일레븐 표면 노출 알부민 결합 사에 관여하지 아미노산, 조합 라이브러리를 생성하는 유전자 무작위를했다. 무작위로 결합 표면 (그림 1) 배열의 소설과 단백질 라이브러리는 파지 디스플레이 기술에 의해 바인더의 선택을 촉진하는 파지 입자에 표현했습니다. dimeric에 대한 biopanning의 여러 라운드를 통해 포도상 구균 단백질 5 HSA와 소설 대상을 6 밝혀졌으며 모두 친화력이있는 작은 bispecific 분자에서 파생된 Z - 도메인. ABDz1로 불리는 소설 단백질 도메인, 다른 분자량, 용해도 및 isoelectric 포인트 대상 단백질의 선택에 대한 정화 태그로 평가되었다. 세 대상 단백질들은 N - 테르에 융합하고 이후 친화력이 정화 소설 태그 Escherishia 대장균으로 표현했다. 고정 HSA 또는 Z - 도메인 중 열을 초기 정화는 relati 결과vely 순수한 제품. bispecific 태그로 2 단계 친화 정화는 단백질 순도의 상당한 개선 결과. Z - 도메인 색층 미디어 확실히 예 MabSelect위한 고정화 항체의 정화를 위해 즉시 사용 가능하며 HSA 쉽게 화학적으로 두 번째 매트릭스를 제공하는 미디어에 결합하실 수 있습니다. 복구 대상 단백질의 순도에 대한 높은 수요가있을 때이 방법은 특히 유리한 것입니다. 표현 호스트의 신진 대사 부담이 태그의 작은 크기로 인해 제한되는 동안 태그의 bifunctionality는 두 가지 색층 단계가 사용할 수 있습니다. 그것은 여러 개의 태그를 조합하여 1,7에 사용 어디에 조합 태그라는 경쟁력있는 대안을 제공합니다.

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

소설과 고효율 두 단계 친화도 크로마 토그래피 프로토콜이 개발되었고 자세하게 설명되어 있습니다. 방법은 두 개의 본질적 동질성과 작은 정화 태그를 기반으로하고 다른 특성을 가진 대상 단백질의 다양한 적용됩니다.

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 ...

orthogonal-protein-purification-facilitated-small-bispecific-affinity

Research

JoVE Journal

Biology

16.9K 조회수.  Royal Institute of Technology. 소설과 고효율 두 단계 친화도 크로마 토그래피 프로토콜이 개발되었고 자세하게 설명되어 있습니다. 방법은 두 개의 본질적 동질성과 작은 정화 태그를 기반으로하고 다른 특성을 가진 대상 단백질의 다양한 적용됩니다.

비디오: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

연구

생물학

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

불안 정한 다중 단백질 단지의 분리에 의해 생체내에 제어 세포 간 연결 및 단백질 자기 동질 크로마 토그래피

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104의 정화, 단백질 Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

선호도 태그 재조합 단백질의 높은 처리량 정화

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

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. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

미세 규모 Thermophoresis에 의한 결합력 결정 단백질 정제 - 무료 방법

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

재조합 단백질의 미끼를 사용 파지 디스플레이와 궁합 선택을위한 프로토콜

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

단백질 - 단백질 상호 작용을 확인<em&gt; 초파리</em탠덤 선호도 정화로&gt; 성인 머리 (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...