우리는 방법의 개발에 선도적 인 토론을위한 앤 마리 디온 - 코트 감사합니다. JK와 RB는 FQNRT 박사 학자이며, M.-MG는 바니 에르 CIHR 학자이며, J.-YM는 FRSQ 수석 연구원이다. 이 작품은 자연 과학 및 JY에 공학 연구 협의회에서 기금에 의해 지원되었다. M.

1. 재조합 배큘로 바이러스의 제조 배큘로 바이러스는 주로 단지 약간의 수정 제조자의 프로토콜에 따라 인비 트로겐의 바크 투 바크 배큘로 바이러스 발현 시스템을 사용하여 생성된다 : <ol><li> 수정 pFastBac1 벡터 (기브, 수명은) GST와 그의 - 태그 (그림 2)를 포함 만들었습니다. PET-52B (+) 벡터 (노바 젠)의를 다중 사이트 (MCS)는 10xHis 태그를 포함, pFastBac1 - 패 혈성-10xHis를 생성하는 pFastBac1 벡터의 MCS에 삽입했다. PET-52B (+) MCS 시퀀스는 5에서 BglII에 제한 사이트 &#39;말단과 3에서 HindIII의 제한 사이트&#39;말단을 추가를 XbaI 및 BlpI 제한 부위 사이에 증폭 된 PCR했다. PCR 생성물을 B​​amHI 및 다음 BglII에 제한 시퀀스의 호환성을 이용하여 제한 효소 BamHI과 HindIII로 pFastBac1의 제한 부위 사이에 클로닝 하였다. 목표는 표현을 허용 pFastBac1를 생성 할 때부터스트렙 타비 딘과 10xHis - 태그 재조합 단백질, PET-52B (+)의 BamHI로 제한 사이트는 MCS 시퀀스는 사용되지 않았다. 또한, XbaI의 제한 사이트는 이미 pFastBac1의 MCS 및 PET-52B의 MCS 삽​​입이 (+)의 사이트 사이에서 발생하는 제한 사이트의 중복을 피하기 위해 사용되지 않았습니다. PreScission 단백질 분해 효소 인식 부위 (LeuGluValLeuPheGln / GlyPro)와 GST 코딩 순서는 다음 pFastBac1 - 패 혈성-10xHis에 삽입했다. 유전자 pGEX-6P-1 (GE 헬스 케어)에서 GST의 cDNA 서열은 PCR 각각 5에서을 NcoI 및 KpnI으로 제한 부위를 함유하는 프라이머 &#39;및 3&#39;말단으로 증폭 하였다. 을 NcoI 사이트는 개시 코돈의 첨가뿐만 아니라 GST에 알라닌을 허용한다. PCR 생성물이어서 스트렙 타비 딘 - 태그의 삭제 결과를 NcoI 및 KpnI으로 제한 부위 사이 pFastBac1-Strep의-10xHis로 클로닝 하였다. 이렇게 얻어진 새로운 융합 벡터 pFastBac1-GST-10xHis 지명되었다. </li><li> 홍보에 대한 cDNA를 코딩을 복제GST (N-말단)과 그의 태그 (C 터미널) 사이의 pFastBac-GST-10xHis 벡터에 관심 otein. C-말단 그의 태그와의 융합을 허용하는 cDNA를 정지 코돈을 제거 할주의하십시오. </li><li> E.에게 변환 (단계 1.2에서 얻은) 재조합 pFastBac와 대장균 DH10Bac는 백 미드를 생성합니다. 식민지 Bluo - 여자와 IPTG (10 ㎍ / ㎖의 테트라 사이클린, 50 ㎍ / ㎖의 카나마이신, 12 ㎍ / ㎖의 겐타 마이신, 100 ㎍ / ㎖의 Bluo을 함유 선택 접시에 30 ° C에서 37 ° C 몇 시간에서 하룻밤 성장 할 수 있도록 허용 여자, 40 ㎍ / ㎖의의 IPTG). </li><li> 선택과 화이트 표현형을 확인하기 위해 선택 접시에 (단계 1.3에서) 2 화이트 식민지를 restreak. </li><li> 10 ㎍ / ㎖의 테트라 사이클린, 50 ㎍ / ㎖의 카나마이신, 10 ㎍ / ㎖의 겐타 마이신 단계 1.4에서 두 개의 흰색 식민지로하고 37 ℃에서 교반하면서 밤새 (250 RPM)를 물게을 포함하는 LB 2 ㎖를 접종 </li><li> 백 미드의 DNA를 정제하기 위해서는, 단계 1.5에서 박테리아 펠렛용액 300 μL 난에있는 세포를 재현 탁 및 솔루션 II 300 μL (퀴아 Maxiprep 키트)를 추가합니다. 실온에서 5 분 동안 품어 3 M KOAc이다 산도 5.5의 300 μl를 추가하고 얼음에 10 분 동안 품어. 4 ° C, 10 분 동안 최대 속도로 샘플을 원심 분리기. 상층 액에 이소프로판올 800 μl를 추가하고 얼음에 10 분 동안 품어. 4 ° C에서 15 분 동안 원심 분리하여 70 % 에탄올 500 μL로 펠렛을 씻으십시오. 펠릿 건조시켜 트리스​​ - EDTA (에틸렌 다이아 민 테트라 초산) 산도 8.0의 40 μL의 멸균 조건을 재현 탁. 백 미드의 DNA는 4 ℃에서 보관해야 </li><li> 제조 업체의 프로토콜에 설명 된대로 배큘로 바이러스를 생성합니다. 배큘로 바이러스는 빛으로부터 보호 4 ° C에 저장할 수 있습니다. </li></ol> 2. 재조합 단백질 발현 <ol><li> 정제를위한 가장 효율적인 배큘 선택할 및 단백질 발현의 피크에 도달 몇시 모니터하기 위해, 미니 감염 분석을 수행의는 다음과 같다 : 2 인 Sf9의 X 10 7 배큘로 바이러스의 133 μL (1 / 150)으로 (10 % FBS, 1 % P / S 보완) 그레이스의 미디어에있는 27 ° C에서 배양 된 세포를 감염 감염하십시오. 0, 24, 48에 1.5 ㎖를 분취를 수집하고, 72 시간 게시물 감염. 펠렛은 세포 및 안티 태그 항체를 사용하여 웨스턴 블롯에 의해 그 단백질의 발현 수준을 분석한다. </li><li> 바이러스와 미디어 사이의 1 / 150의 비율로 미디어 500 ㎖에 ML 당 1.0 × 10 6 인 Sf9 세포를 포함하는 회 전자를 감염시키기 위해 2.1 단계에서 가장 효율적인 배큘로 바이러스를 사용합니다. 감염된 세포는 27 ° C에서 서스펜션의 성장과 최대의 단백질 발현 (위 2.1 단계에서 결정)에 도달하면 세포를 수확 할 수 있습니다. 펠렛 세포는 나중에 사용할 때까지 -80 ° C에 저장할 수 있습니다. </li></ol> 3. 수용성 세포 용 해물의 제조 다음 배양 단계는 모두 가벼운 회전에서 4 ° C에서 수행됩니다. <ol><li> 를 Lyse SGST 결합 완충액 (150 mM의 염화나트륨, 1 mM의 EDTA, 0.05 % 트리톤 - X-100, 1 ㎜ 250 ㎜의 염화나트륨의 최종 농도에 대한 PBS1X의 DTT, 및 단백질 분해 효소 억제제 ~ 20 ㎖에 관심 단백질을 발현 F9 세포 칵테일 (로슈). 빙수 욕에서, 다운스 균질 기, 초음파 처리 3X 30초 각 (70 % 출력)와 용액 20X 다운스 다시 20X를 다운스. </li><li> (선택 사항). DNA 또는 RNA의 오염을 제거하기 위해 4 ° C에서 30 분 동안 1 mM의 MgCl2를 2 7 클레아 제 (2.5 U / ㎖) 벤조 전체 세포 용 해물을 품어. </li><li> 4 ℃에서 30 분간 18,000 rpm으로 원심 분리기 뜨는을 유지합니다. </li><li> (선택 사항). 원심 분리기는 상층 액을 다시 4 ° C에서 18,000 rpm으로 30 분 동안 명확한 가용 해물를 얻을 수 있습니다. 막힘을 방지하기 위해 0.2 또는 0.45 μm의 필터를 통해 뜨는을 전달합니다. </li></ol> 4. GST 구슬에 단백질의 결합 <ol><li> GST 비즈 1 ㎖로 용해 세포 용 해물을 품어 (로 2 회 prewashedGST의 10 ㎖의 부드러운 회전에서 4 ° C에서 1 시간 동안 버퍼)를 결합. </li></ol> 의견 배양 시간은 단백질의 안정성과 결합 효율에 따라 최적화 될 필요가있다. <ol start="2"><li> 빠른 700 rpm에서 스핀과 결합되지 않은 단백질을 함유 뜨는을 제거 (이는 추가 분석을 위해 유지 될 수있다). GST 세척 버퍼 GST-결합 단백질 (그림 3B, 레인 1) 세척 (GST는 350 mM의 염화나트륨 마지막으로 버퍼를 바인딩). </li><li> 단계를 반복 4.2 배. 마지막 세척에서, 가능한 한 많은 뜨는을 제거합니다. </li><li> 열 충격 단백질 (8)의 비특이적 결합을 방지하기 위해 4 ° C에서 1 시간 동안 (10 ㎖ GST 바인딩 버퍼) 5 mM의 ATP, 15 mM의 MgCl2를 가진 구슬을 품어. GST 세척 버퍼로 구슬을 세 번 씻으십시오. </li></ol> 5. GST의 PreScission 분열 <ol><li> GST 비즈를 원심 분리기, 뜨는을 제거하고 P5 버피와 GST 비즈를 씻어FER (50 mM의 NaHPO 4의 pH 7.0, 500 mM의 NaCl을, 10 % 글리세롤, 0.05 % 트리톤 - X-100, 5 mM의 이미 다졸). </li><li> 3 시간에 밤새 4 ℃에서 P5 버퍼 PreScission 효소와 GST-구슬을 품어 (100 μL의 분수 GST 비즈를 나누고 P5 버퍼 100 ㎕의 희석 효소의 4-8 단위 (2-4 μL)를 추가). </li></ol> 의견 PreScission 단계의 배양 시간은 분자량뿐만 아니라 단백질의 안정성에 따라 최적화 될 필요가있다. 열화가 관찰되는 경우,이 배양 시간 밤새 2 시간의 최소 대신에 감소 될 수있다. <ol start="3"><li> 빠른 스핀과 상층 액을 수집합니다. P5의 100 μl를 추가 한 후이 단계를 두 번 반복 구슬에 버퍼 및 모든 분수 (그림 3B, 레인 2) 풀. </li></ol> 코멘트 : 나머지 구슬 절단 효율성의 분석을 위해 사용될 수있다 (그림 3B </stroNG&gt;, 레인 3). 일반적으로, 단백질 70 ~ 80 %가 절단된다. 6. 단백질 결합 TALON 금속 친 화성 수지에 <ol><li> 1 ㎖의 3 분수에 위의 용출을 나누고 회전에서 1 시간 동안 (P5 버퍼로 2 회 prewashed) 100 ㎕의 발톱 금속 친 화성 수지 건조 구슬과 각을 품어. </li></ol> 코멘트 : 여러 분수 용출을 나누면 바인딩 / 세척 효율을 증가시킨다. <ol start="2"><li> P30 버퍼 (30 mM의 이미 다졸의 최종 농도로 P5 버퍼)와 수지 5 분을 씻으십시오. </li><li> 단계 6.2 배를 반복하여 하나의 튜브에 비즈를 풀. 마지막 세척하기 전에 가능한 한 많은 뜨는을 제거합니다. </li></ol> 코멘트 :이 TALON 바인딩 샘플 (그림 3B, 4 레인)에 해당합니다. 7. 정제 된 단백질의 용출 <ol><li> P5와 회전에서 5 분의 단백질 결합 TALON 수지를 품어00 버퍼 (500 MM의 이미 다졸의 최종 농도로 P5 버퍼). 버퍼 1:1 V / V의 구슬의 비율을 사용 (당신이 건조 구슬 200 μl를 촬영 한 경우 예를 들어, 당신은 P500의 200 μl를 추가해야합니다). 이 단계를 두 번 반복하고 개별적으로 각각의 용출 분획을 유지 (, 그림 (b), 5-7 차선), 용출 1 (E1)라는 이름의 용출 2 (E2 및 용출 3 (E3)). </li></ol> 의견 남은 비즈 용출 효율 (도 3b, 레인 8)의 분석에 이용 될 수있다. 전형적으로, 단백질 60 ~ 80 %가 전체에 용출된다. <ol start="2"><li> 쿠마시 블루 (그림 3B) 또는 시각화를위한 SYPRO 단백질 얼룩 SDS-PAGE와 얼룩에 표준 BSA 농도가 각 부분의 약 20 ~ 40 μl를로드하여 정제 된 단백질의 양과 질을 분석합니다. </li></ol> 코멘트 : 관심의 단백질은 또한 죽 검출 할 수있다항-GST 및 안티 그의 항체 (도 3C)를 이용하여 정제 절차 ughout. 8. 정제 된 단백질의 저장 <ol><li> 4 ℃에서 적절한 저장 버퍼에 대한 정제 된 단백질 (예를 들어, 20 mM 트리스 아세테이트 산도 8.0, 200 MM의 한국 공항 공사, 10 % 글리세롤, 1 mM의 EDTA, 0.5 mM의 DTT)을 Dialyze 그것은 투석 중에 정제 된 단백질의 침전을 확인하는 것이 중요합니다. 우리는 일반적으로 교반 회전에서 4 ° C에서 1 시간 동안 2 배 dialyze. </li><li> 정제 된 단백질 (10 ~ 20 μL)의 작은 분취 량을 확인하고 30 분 동안 드라이 아이스에 냉동. -80 ° C에서 분취를 저장하고 여러 해동 / 냉동 사이클을 피할 수 있습니다. </li></ol>

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여기에 제시된 GST - 그의 정화 프로토콜은 재조합 단백질의 크기의 넓은 범위의 정화를 위해 적당하다 : 우리는 성공적으로 리 RAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa), 불안정한 고 분자량의 단백질을 정제 리 슈만 편모충의 infantum : 리튬 BRCA2 (125kDa) 6.9. 또한,이 프로토콜을 사용하여 정제 된 단백질은 6 생화학 적 활성했다. 이 방법의 성공은 전적으로 목적 단백질...

재조합 단백질의 정제는 생화학의 근본적인 문제를 해결하기 위해 매우 중요합니다. 이온 교환 크로마토 그래피 및 크기 배제 크로마토 그래피와 같은 단백질 정제의 통상적 인 방법은 각각 같은 그것의 등전점과 충전 또는 크기와 같은 표적 단백질의 물리적 특성에 의존한다. 후자 단백질 특성은 상당히 통상적 인 단백질 정제 전략 단백질 오염의 기회를 증가 단백질의 다양성에 의해 공유된다. 이 문제는 시간 소모적이다 체류 정화 컬럼의 사용으로 피할 수있다. 동시에, 후자의 크로마토 그래피 방법은 고가의 실험 구성을 요구한다. 대부분의 경우 태그가 관심의 단백질에 고유하므로 선호도 태그 정화가 강하게 대상 특이성을 증가시킨다. 최근 연구에서, 플래그 또는 HA-친화력 정화 널리 사용되어왔다. 기존 REC 대조적하나의 태그가 사용되는 ombinant 단백질 정제 프로토콜은, 우리는이 태그의 고유 한 조합을 설립. 우리의 방법은 양 원하는 단백질의 순도 사이의 최적의 비율에 대한 관심의 단백질의 C-말단의 N-말단과 그의 태그에서 GST-태그의 융합을 포함한다. GST는 글루타티온 세 파로스 구슬 정화에 매우 효율적이다 긴 태그 (29 kDa의)입니다. 또한, GST를 사용하여 우리의 방법 1의 비용 효율성을 보장합니다. (두 아미노산의 첨가의 결과로 인식 서열 LeuGluValLeuPheGln / GlyPro 가진) PreScission 효소와 GST를 절단 할 수있는 가능성은 많은 장점을 가지고 예를 들어, 이러한 전략은 알로 스테 릭 장애물로 인해 생리적 단백질 기능의 변경을 방지한다. 다른 단백질 말단에 융합 작은 그의 태그는 벽개 GST를 씻어 내고,뿐만 아니라 분해 단백질 및 기타에 의해 단백질 순도를 증가시키기 위해 제 정제 공정에 제공<html> 오염 물질. 그의 정화 단계의 열 (TALON 수지)에 GST 전환 할 때 또한 프로토콜은 투석 단계가 필요하지 않습니다. 이러한 정제 과정에서 일반 오염 물질은 열 충격 단백질 (HSP)이다. MgCl2를 ATP와 함께 인큐베이션 단계의 첨가는 이러한 오염물을 제거 (도 1)을 허용한다. 고속 단백질 액체 크로마토 그래피 (FPLC) 및 고성능 액체 크로마토 그래피 (HPLC)는 관심의 정제 된 단백질의 높은 수율을 생성하기 위해 고가의 장비에 의존하는 일반적인 기술이다. 우리가 여기에 존재하는 배치 정화 프로토콜은, 대조적으로, 수동이며, 고가의 악기 설정을 필요로하지 않습니다. 단백질의 높은 수율이 프로토콜을 확장하여 도달 할 수 있습니다. 동시에 일괄 정제 프로토콜, 용출 부피는 FPLC 또는 HPLC와 관련되지 않은 단백질의 농도를 향상시키기 위해 조정될 수있다. ntent &quot;&gt; 또, 일괄 GST-그의 정제 프로토콜 ~ 10-300 kDa의의 크기 범위를 가진 단백질을 정제 할 수있다. 큰 장점 중 하나는 하나가 동시에 여러 단백질을 정제 할 수 있다는 사실에 의해 주어진다 예를 들어, 야생형과 돌연변이 단백질 둘. 제시된 프로토콜의 성공은 전적으로 그 단백질의 발현 수준과 용해도에 의존한다.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

gst-his purification: a two-step affinity purification protocol yielding full-length purified proteins

체외에서 단백질의 생화학 적 특성에 대한 효소 학의 핵심 분석은 그 정제 된 단백질의 높은 농도를 필요로. 단백질 정제 프로토콜은 효율, 단순성 및 비용 효율성을 배합 한 것이다. 여기에서, 우리는 N-말단 글루타티온 세 파로스 태그 (GST) 2,3 모두 융합 C-말단 10xHis 태그 4를 기반으로 재조합 단백질에 대한 새로운 소규모의 친화력 정화 시스템으로 GST - 그의 방법을 설명 관심의 단백질. 후자의 구조는 단백질 발현을위한 5 인 Sf9 감염된 세포의 감염을 위해, 배큘로 바이러스를 생성하는데 사용된다. GST는 정화 효율을 보장하는 역할을 다소 긴 태그 (29 kDa의)입니다. 그러나, 단백질의 생리 학적 성질에 영향을 미칠 수있다. 따라서, 후속 적으로 10 PreScission 효소를 사용하여 단백질을 분해한다. 최대 순도를 보장하고, 쪼개진 GST를 제거하기 위해 우리비교적 작은 그의 - 태그를 기반으로 두 번째 친 화성 정제 단계를 추가했습니다. 중요한 사실은, 우리의 기술이 따라서 저하 된 단백질을 제거하기위한 효과적인 도구이다 단백질의 양단 측면에 위치하고있는 두 개의 서로 다른 태그에 기초하고, 전장 단백질을 풍부. 여기에 제시된 방법은 FPLC 등 고가의 악기 설정을 필요로하지 않습니다. 또한, 우리는 열 충격 단백질 불순물과 핵산을 오염 폐지하는 핵산 분해 효소 처리를 제거하는 MgCl2를하고 ATP 세척을 통합. 요약하면, 플 랭킹이 다른 태그의 조합을 N-및 C-말단 및 태그 중 하나를 절단하는 기능을, 또한 고순도 및 전장 단백질의 회수를 보증한다.

GST-그의 정제 프로토콜의 효율을 예시하기 위해, 우리는 Rec14, S. 정제 32.9 kDa의의의 pombe 단백질. Rec14 cDNA를 각각 N-및 C-말단 (그림 1A)에서 GST - 그의 - 태그의 추가를 허용하는 우리의 수정 pFastBac1 벡터에 클로닝 하였다. 재조합 배큘로 바이러스는 다음 제조 및 단백질 발현을 위해 감염된 SF9 세포를 감염시키기 위해 사용되었다. 수용성 세포 용 해물은 GST 비즈 배양과 결합...

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

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

본 프로토콜에서는, 고유 분해 가능한 GST-태그 및 그의 작은 태그를 조합하여 재조합 단백질의 정제를 허용하는 매우 효율적이고 비용 효과적인 소량의 단백질 정제 방법을 보여준다.

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. ...

gst-his-purification-two-step-affinity-purification-protocol-yielding

Research

JoVE Journal

Biology

41.3K 조회수.  Hôtel-Dieu de Québec.  본 프로토콜에서는, 고유 분해 가능한 GST-태그 및 그의 작은 태그를 조합하여 재조합 단백질의 정제를 허용하는 매우 효율적이고 비용 효과적인 소량의 단백질 정제 방법을 보여준다. 

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

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

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

높은 활성 효모 변이의 색층 정화

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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 ...

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 ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

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 ...

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. ...