이 작품은 NSERC 부여에 의해 지원되었다 386634. 우리는 도움이 토론 Screaton 연구소의 회원들에게 감사의 말씀을 전합니다.

시약, 플레이트, 및 세포의 1. 준비 <ol><li> 25 mM 트리스 pH가 7.5, 150 mM의 염화나트륨, 50 mM의 불화 나트륨, 0.5 mM의 EDTA pH를 8.0, 0.5 % 트리톤-100, 5mM의 베타 - 글리세로 포스페이트, 5 % 글리세롤 : 500 ml의 용해 완충액을 만든다. 4 ℃에서 보관하십시오. 사용 1 mM의 디티 오 트레이 톨 (DTT), 1 mM의 페닐 메틸 설 포닐 플루오 라이드 (PMSF), 및 1 mM의 나트륨 바나 데이트를 추가하기 직전. 이 단계 후에, PMSF가 임의의 세정 완충액으로 요구되지 않는다. </li><li> 200 mM 트리스의 pH 7.5, 50 mM의 베타 글리세 (FW 216), 2 mM의 나트륨 바나 데이트 : 10 배 키나제 버퍼의 20 mL로한다. -20 ° C에서 1 ml의 튜브에 나누어지는 및 저장. 사용 5 mM의 DTT를 추가하기 직전. </li><li> 300 μM 아데노신 삼인산 (ATP), 66 밀리미터의 MgCl 2, -20 ° C에서 33 mM의 MnCl 2 1 ml의 튜브에 나누어 저장 : 10 배 M-ATP 버퍼의 20 mL로한다. </li><li> 배 도데 실 황산나트륨 100 mL로 (SDS)를 용해 완충액 : 1.5 g 트리스베이스, 글리세롤 20 ㎖, 30 ㎖의 H 2 O. 용해 및 HCl로 6.8에 산도를 조정합니다. 40 추가ml의 10 % SDS는 100 ㎖에 볼륨을 조정합니다. 25 mg의 브로 모 페놀 블루를 추가합니다. </li><li> 스팟 따라 96 웰 플레이트와 라벨 플레이트 세트에서 잘 당 GST-키나제 (14)를 인코딩 (25) NG / μL 포유 동물 발현 플라스미드의 4 μL. 사용할 때까지 -20 ° C에서 인감 및 동결 판. </li><li> 형질 전환 1-2 일 전에, CO 2 (최종 5 %)로 보충 된 37 ℃ 배양기에서 배양 HEK293T의 완전한 둘 베코 변성 이글 배지 (DMEM) + 10 % 소 태아 혈청 (FBS)과 항생 물질의 세포. 트립신과 통로는 주식이 확장시&gt; 80 % 합류가 될 수 없습니다. 6 × 10 7 세포의 최소 화면 (80 % 합류에 HEK293T 세포의 약 3 × 15cm 요리)가 필요합니다. </li></ol> 2. 형질 참고 : 전체 프로토콜의 흐름도는 그림 1을 참조하십시오. <ol><li> 키나아제 플라스미드를 함유하는 플레이트를 동결 되었으면, 실온에서 해동차 우물의 바닥에서 습기를 수집하기 위해 3 분 동안 1,900 XG에 원심 분리기. </li><li> 지질 기반 형질 전환 시약의 312.7 μL와 감소 혈청 배지 (예를 들어, OPTI-MEM)의 8.6 ml의 혼합. 5 분 동안 앉아 보자. </li><li> 작은 볼륨 카세트를 장착하는 자동화 된 액체 디스펜서를 사용하여 각 웰에 감소 혈청 매체의 10 μl를 추가합니다. </li><li> 작은 볼륨 카세트를 장착하는 자동화 된 액체 디스펜서를 사용 단계 2.2에서 감소 된 혈청 배지 / 형질 전환 시약 믹스 잘 당 10 μl를 추가합니다. 20-45 분 동안 앉아 보자. </li><li> 전체 80ml의 DMEM에 7.5 × 105 세포 / ml로 재현 탁 293T 세포. 잘 표준 볼륨 카세트 ​​장착 자동 액체 디스펜서를 사용하여 당 세포 현탁액 (즉, 7.5 × 10 4 세포)의 100 μl를 추가합니다. </li><li> 균일 한 세포 분포에 대한 현미경으로 우물을 확인하고 24 시간 동안 인큐베이터로 돌아갑니다. </li></ol> 3. GST-키나제 풀다운 <ol><li> F 확인resh : H 2 O의 540 μL와 0.2 M 나트륨 바나 데이트 60 μl를 혼합하여 4 mM의 pervanadate 솔루션 제 2 튜브 믹스 30 % 과산화수소 2.7 μL와 PBS 1.4 ml 중에. 함께 두 가지 솔루션을 추가하고 사용하기 전에 15 분 동안 앉아 보자. </li><li> (이것은 또한 너무 많은 난류를 생성로서 자동화 액체 디스펜서를 사용하지 않는) 단계 3.1에서 제조 pervanadate 2.5 μL,이어서 각 웰에 0.25 M CaCl2를 2 μL 분주 멀티 채널 피펫을 사용. 10 분 동안 37 ℃에서 각각의 판을 품어 후 얼음에 배치합니다. </li><li> 제 1 (시약의 제조)에 나타낸 바와 같이 DTT, PMSF, 나트륨 바나 데이트를 첨가하여 용해 완충액 35 mL로 제조. </li><li> 얼음에 판을 유지, 각 웰 진공 흡입기를 사용하는 매체를 제거합니다. 즉시 표준 카세트를 장착하는 자동화 된 액체 디스펜서를 사용하여 50 μL / 웰의 차가운 얼음 용해 버퍼를 추가합니다. 를 Lyse에 얼음에 30 분 앉아 보자 (옵션 : 작은지면을 밀봉하여 여기에 필요한 경우 중지 할 수 있습니다전자 및 -80 ° C에서 저장. 얼음에, 해동 판)을 계속합니다. </li><li> 4 ℃에서 3 분 동안 1,900 XG에 스핀 판. </li><li> 각 웰 멀티 채널 피펫을 사용하는 세포를 긁고 모든 내용을 전송 적절 V 바닥 96 웰 플레이트를 표시합니다. 4 ℃에서 10 분 동안 1,900 XG에 스핀 판. </li><li> 회전하는 동안, 린스 100 μL / 웰의 (PMSF)없이 차가운 얼음 용해 버퍼와 글루타티온 코팅 된 플레이트 (각 96 웰 플레이트에 대해 하나)를 입력합니다. 얼음에 판을 보관하십시오. </li><li> 원심 분리기에서 V-바닥 플레이트를 제거합니다. 한 번에 하나는 종이 타월에 용해 버퍼와 얼룩을 흔들 싱크를 통해 글루타티온 판을 반전. 하단에있는 펠렛을 방해하지에주의하면서 판을 기울 멀티 채널 피펫을 사용하여 글루타티온 판에 V-바닥 판에서 용해 버퍼를 전송합니다. 판 커버와 결합하는 최소 2 시간 동안 얼음에 둡니다. </li><li> 2 시간 바인딩 단계의 마지막에 가까운, T을 보장 방사능 워크 스테이션을 준비그는 필요한 안전 조치는 방사성 작업 장소에 있습니다. 30 ° C로 설정 하이브리드 오븐. </li><li> 이 단계에서 필요하지 않은 제 1 절 PMSF에 표시된 추가 DTT 나트륨 바나 데이트에 의해 용해 완충액 200 ㎖를 준비합니다. </li><li> 용해 버퍼를 흔들 종이 타월에 가릴 수있는 싱크대 글루타티온 플레이트 전환. 헹구고 웰 (PMSF)없이 100 ㎕의 용해 완충액으로 3 배. 진행 할 준비가 될 때까지 세정에 보관 - 우물이 마른 앉게하지 마십시오. </li><li> 10 배 주식을 희석하고 표준 볼륨 카세트를 장착하는 자동화 된 액체 디스펜서를 사용하여 1X KB의 50 μL 1. 린스 판 한 번 섹션에 표시된대로 DTT를 추가하여 1X 키나제 버퍼 (KB) 55 mL로 준비합니다. 솔루션이 준비가 될 때까지 우물에 1X KB를 남겨주세요 : <ol><li> 15.9 ml의 최대 530 관심있는 기판의 μg의, 미엘린 염기성 단백질 500 μg의 (MBP), 10 배의 2.65 ml의 KB, 1 M DTT의 13.25 μL, 및 H 2 O 500을 추가하여 솔루션을 준비합니다. </li></ol></리&gt; <li> 한 번에 하나씩, 1X KB 린스를 제거 종이 수건에 오점, 바로 작은 볼륨 카세트를 장착하는 자동화 된 액체 디스펜서를 사용하는 솔루션의 30 μl를 추가 싱크대 위에 접시를 반전. 얼음에 판을 보관하십시오. </li><li> 2.5 ml의 10 배 M-ATP, 500 μCi를 감마 (32) P의 ATP, 및 H 2 O에 10 ㎖를 첨가하여 방사능 작업 영역 용액 B를 준비한다. <ol><li> 잘 배출 힘에 의한 혼합에 도움 리피터 피펫을 사용하여 당 용액 B 20 μl를 추가합니다. 덮고, 30 분 동안 30 ° C의 오븐에서 혼성화 부화. </li></ol></li><li> 30 분 후, 얼음에 다시 접시를 전송합니다. 각 웰 멀티 채널 피펫을 사용하여 2 배 SDS 용해 버퍼의 50 μl를 추가합니다. 다음 단계로이 시점에서 진행, 또는 편리한 때까지 -20 ℃에서 알루미늄 호일 및 저장과 번호판을 봉인 할 수 있습니다. </li></ol> 4. 달리기, 염색, 건조 젤 참고 : 모든 작업은 지역 DESIGNA에서 수행되어야한다방사능에 대한 테드. <ol><li> 하이브리드 오븐의 전원을 켜고 85 ℃로 설정합니다. 실온에서 해동 판. 일단 오븐 오븐 10 분 샘플을 변성하는 동안 배양 온도, 이전 판에 도달했습니다. </li><li> 멀티 채널 피펫을 사용하여 각 반응의 15 μL와로드 26 웰 프리 캐스트 젤은 한 번에 여러 우물을 채우기 위해. 케어는 모든 팁 대응도 전에 샘플을 추가로 정렬 않도록주의해야합니다. 이 비법 인 ATP를 포함로 추적 염료 (파란 선)은 젤의 바닥 떨어져 실행하지 못하게 150 V.에서 젤을 실행합니다. </li><li> 젤을 해체하고 메스하거나 영화를 과다하므로 직선을 사용하여 비법 인 ATP (파란 선)을 차단. 라벨이 붙은 용기에 젤을 놓고 15 분 동안 쿠마 얼룩 커버. </li><li> 간단히, 쿠마 얼룩을 제거 물 젤을 씻어 및 솔루션을 destain 추가합니다. 젤 Destain 단백질은 명확하게 볼 수 있습니다 때까지. MBP를위한 밴드 및 기판 밴드해야각 샘플에 대한 표시. </li><li> 젤을 건조 : <ol><li> 필터 큰 종이를 잘라 건조기에 놓습니다. </li><li> 이 부드러운 때까지 증류수에 셀로판 시트를 적시고 무료 주름, 용지의 상단에 배치. </li><li> 젤의 순서를 기록하고, 셀로판 시트의 상단에 젤을 놓습니다. 두 번째 셀로판 시트를 적시고 젤 위에 놓습니다. </li><li> 좋은 균일 한 표면에 대한 모든 거품을 (플레이트 밀봉 롤러이 잘 작동) 출시. 플랩을 닫고 진공 켜고 80 ℃에서 3 시간 동안 젤을 건조. </li></ol></li><li> 겔이 건조되면, 신호를 강화하기 위해 스크린을 사용 XAR 필름에 노출. 사란 랩 또는 비닐 봉지와 카세트를 감싸고 서리을 유지하기 위해 테이프로 밀봉. -80 ° C 하룻밤에 카세트를 저장합니다. </li></ol> 5. XAR 필름 개발 <ol><li> 다음 날, 냉장고에서 카세트를 제거하고 실온에서 해동을 할 수 있습니다. 의 필름 개발암실은 제조자의 지시에 따른 필름을 사용하여 프로세서. 참고 : 키나제 기판 쌍의 증거에 대한 영화를 검사합니다. 두 번째 이상 노출도 약한 인산화 이벤트를 감지하는 데 유용 할 수 있습니다. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+kinomics+to+delineate+signaling+pathways:+control+of+CRTC2/TORC2+by+the+AMPK+family.">Using kinomics to delineate signaling pathways: control of CRTC2/TORC2 by the AMPK family.</a> Cell Cycle. 7, 3823-3828 (2008).</li><li>Abu-Thuraia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axl+phosphorylates+elmo+scaffold+proteins+to+promote+rac+activation+and+cell+invasion.">Axl phosphorylates elmo scaffold proteins to promote rac activation and cell invasion.</a> Mol Cell Biol. 35, 76-87 (2015).</li><li>Manning, G., Whyte, D. B., Martinez, R., Hunter, T., Sudarsanam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+complement+of+the+human+genome.">The protein kinase complement of the human genome.</a> Science. 298, 1912-1934 (2002).</li></ol>

The authors have nothing to disclose.

14,15 접근법을 설명 원래 공보 이후, 180 GST-키나제의 원래 라이브러리는 인간 단백질 kinome 420 부재, 또는 ~ 80 %로 확대되었다. 확장 된 라이브러리와 같은 설명 프로토콜 (필요하다고 인정하는) phosphorimaging 및 디지털 신호 향상의 사용에 의해 단축 될 수있는 필름을 개발 4~5일 다음 1~4일 걸립니다. 주의를 기울여야합니다 몇 가지 주요 단계 (프로토콜의 개요를 그림 1 참조)이 있...

단백질 번역 후 변형 (PTMS)는 세포 내 통신을위한 필수적이다. 아마도 모든 PTMS의 연구 최고 세포 내 현지화, 형태, 안정성, 생화학 적 활동을 포함한 단백질 기능의 무수를 조절 단백질 인산화 효소에 의해 촉매 인산화이다. 표적 단백질의 인산화 부위의 식별은 트립신 포스 포 매핑 또는 인산화 펩티드 1,2- 위해 농축 된 샘플을 이용하여 현재 표준 프로테옴 기법에 의해 달성 될 수있다. 발현 프로테옴 사분의 삼가 3 인산화 될 것으로 예상하고 100 만 6까지 예상치 200,000 인산화 부위 5를 식별하는 동안, 이들의 대부분은 통로 또는 단백질 키나제 시그널링 어떠한 할당 생물학 없다. 인산화 사이트의 식별은 비교적 큰 도전이고, 비교적 간단하지만기판 쌍 :이 사이트를 대상으로 동족 키나제 (들), 우리가 매핑 키나제로 참조하는 프로세스를 식별합니다. 기판 쌍이 설명되었지만, 어느 주목 키나아제로 시작하고 그 기판을 찾고 관심 또는 기판에서 시작하여 개질 키나아제 7-11 또는 실험적 계산 (12)을 찾기 위해 시도 : 키나아제를 식별하기위한 여러 가지 방법. 공지 인산화 기판 키나제를 식별하는, 생물 정보학 기판과 강수량 복합체를 형성 키나제 인산화 잔사 (컨센서스 사이트) 측면뿐만 아니라 식별 아미노산의 짧은 보존 된 서열을 포함하는 단백질을 식별하는 데 사용될 수있다. 그러나, 이러한 방법은 시간이 많이 소요되어 종종 성공을 충족하지 않습니다. 우리는 주어진 빠르게 기판 (13)을 인산화 키나아제를 식별 체계적인 접근 기능을 개발 하였다. 스크린 분석법 우수한 특정을 생산잠재적 인 동족 키나제에 대한 아주 명확한 선택과 성만. 생물학적 신호에 인산화의 중심성을 감안할 때, 스크린은 거의 모든 세포 신호 전달 경로 14-16에서 발견에 유용합니다. 스크린은 인간 단백질 키나제의 라이브러리로 대규모 키나아제 분석을 수행하는 것을 포함한다. 키나아제는 박테리아 글루타치온 S 트랜스퍼 라제 (GST) 단백질로 태그 된 재조합 효소 즉, 포유 동물 세포 추출물로부터 정제 - 박테리아로부터 제조 된 것과 달리 - 종종 필요한 상류 단백질 키나제의 존재하에 생성되고 재조합 효소는 시험 관내에서 활성을 갖는. 세린, 트레오닌 및 티로신 키나제 활성이 하류 키나제 활성화에 필요하면서 실제로 효모 (10)에 존재하는, 효모 게놈은 500 유전자 17 포유류 kinome는,하기 위해 훨씬 더 복잡해질 것을 나타내는, 122 단백질 키나제를 코딩 REGULA고차 생물에 고유의 프로세스를 테. 또한, 세포 생물학 및 (등과 소분자, 성장 인자, 호르몬 등) 인간의 질병과 관련된 다른 자극의 효과는 적절한 컨텍스트 (14, 15)는 조절 키나제 활성에 사용될 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lysis buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.1</td>
		</tr>
		<tr>
			<td>10x kinase buffer</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.2</td>
		</tr>
		<tr>
			<td>10x M-ATP</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.3</td>
		</tr>
		<tr>
			<td>Human kinase plasmids</td>
			<td>Orfeome, Invitrogen, Origene</td>
			<td></td>
			<td>GST-tagged in house</td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Fisher Scientific</td>
			<td>CS003595</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH3002201</td>
			<td>supplement with 100U/ml penicillin, 100ug/ml streptomycin, 10% fetal calf serum.</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture dishes</td>
			<td>Fisher Scientific</td>
			<td>877224</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Invitrogen</td>
			<td>22600-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated liquid dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>24073295</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard cassette attachment</td>
			<td>Thermo Scientific</td>
			<td>14072670</td>
			<td></td>
		</tr>
		<tr>
			<td>4mM pervanadate</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 3.1</td>
		</tr>
		<tr>
			<td>0.25 M CaCl2</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20-200 uL)</td>
			<td>Labnet</td>
			<td>p4812-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (1-10 uL)</td>
			<td>Thermo Scientific</td>
			<td>4661040</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 6-well plates</td>
			<td>Evergreen Scientific</td>
			<td>290-8116-01V</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione coated 96-well plates</td>
			<td>Fisher Scientific</td>
			<td>PI-15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Biostad</td>
			<td>350355</td>
			<td></td>
		</tr>
		<tr>
			<td>GST tagged substrate</td>
			<td>Made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin Basic Protein (MBP)</td>
			<td>Sigma</td>
			<td>M1891</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeater pipette (1 mL)</td>
			<td>Eppendorf</td>
			<td>22266209</td>
			<td></td>
		</tr>
		<tr>
			<td>32P gamma-ATP</td>
			<td>Perkin Elmer</td>
			<td>BLU502Z500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>2X SDS lysis buffer (100 mL)</td>
			<td>Made in house</td>
			<td></td>
			<td>See Protocol step 1.4</td>
		</tr>
		<tr>
			<td>26-well precast TGX gels</td>
			<td>BioRad</td>
			<td>567-1045</td>
			<td>gel percentage required is dependent on the molecular weight of the substrate of interest</td>
		</tr>
		<tr>
			<td>Coomassie stain</td>
			<td>Made in house</td>
			<td></td>
			<td>0.1% Coomassie R250, 10% acetic acid, 40% methanol</td>
		</tr>
		<tr>
			<td>Coomassie destain</td>
			<td>Made in house</td>
			<td></td>
			<td>10% acetic acid, 20% methanol</td>
		</tr>
		<tr>
			<td>Labeled gel containers</td>
			<td>Made in house</td>
			<td></td>
			<td>Used plastic lids from empty tip boxes, just big enough to contain one gel</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>Fisher Scientific</td>
			<td>57144</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellophane sheets (2)</td>
			<td>BioRad</td>
			<td>165-0963</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel dryer</td>
			<td>Labconco</td>
			<td>4330150</td>
			<td></td>
		</tr>
		<tr>
			<td>Double emulsion autoradiography film</td>
			<td>VWR</td>
			<td>IB1651454</td>
			<td></td>
		</tr>
		<tr>
			<td>Film cassette</td>
			<td>Fisher Scientific</td>
			<td>FBAC-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Intensifying screen</td>
			<td>Fisher Scientific</td>
			<td>FBIS-1417</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealing rubber roller</td>
			<td>Sigma</td>
			<td>R1275</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of kinase-substrate pairs using high throughput screening

우리는 새로운 신호 전달 경로를 밝히기 위해 사용될 수 인산화 기판 전용 인간 단백질 키나제를 식별하기위한 스크리닝 플랫폼을 개발했다. 우리의 접근 방식은 정제 GST - 태그 인간의 단백질 키나아제의 라이브러리와 관심의 재조합 단백질 기판의 사용을 특징으로한다. 우리는, CREB-규제 전사 보조 활성화 인자 2 (CRTC2)에 글루코스 조절 사이트 키나아제 MAP / 미세 소관 연관 조절 키나제 2 (MARK2)를 식별하는 베타 세포의 증식에 필요한 단백질뿐만 아니라을이 기술을 사용한 티로신의 액슬 제품군은 어댑터 단백질 엘모의 인산화에 의한 세포 전이의 규제로 키나아제. 우리는이 기술을 설명하고 세포가 환경 자극에 반응하는 방법의 포괄적 인지도를 구축하는 데 도움이 될 수 있습니다 방법에 대해 설명합니다.

화면에서 대표적인 결과는도 2에 도시되어있다. 180 키나아제는 CRTC2뿐만 아니라 고전 키나아제 검정 기질 미엘린 염기성 단백질 (MBP)로부터 AA 2백68부터 2백83까지 대응 GST 표지 된 펩티드 기질을 사용하여 스크리닝 하였다. 단 두 키나제, MARK2와 매우 관련 키나아제 MARK3는 CRTC2 펩타이드를 인산화. 그것은 많은 phosphorylatable 잔류 물을 포함하고 젤의 아래쪽으로, 18 kDa의에서 실행되는 MBP,...

Watch this Scientific Journal Video about Identification of Kinase-substrate Pairs Using High Throughput Screening at JoVE.com

Identification of Kinase-substrate Pairs Using High Throughput Screening

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

identification-kinase-substrate-pairs-using-high-throughput

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JoVE Journal

Biology

8.1K 조회수.  Children's Hospital of Eastern Ontario Research Institute. Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

비디오: Identification of Kinase-substrate Pairs Using High Throughput Screening

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

형광등과 높은 처리량 검사 및 Biosensing C. 엘레간스 레인스

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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생물학

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

에서 곰팡이 Endoglucanase 활동의 높은 처리량 검사 대장균</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

자동을 사용하여 염색체의 높은 처리량 물리적 매핑<em&gt; 현장에서</em&gt; 하이브리드화

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

자동화 모​​듈 높은 처리량 엑소 폴리 사카 라이드 심사 플랫폼은 매우 민감한 탄수화물 지문 분석과 결합

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

멀티 효소 높은 처리량 유전자 효소 검사 시스템을 사용하여 상영

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...