이 연구는이 지원되었습니다 오스트리아 과학 재단 (FWF), 유럽위원회 (European Commission) 통합 프로젝트 MiMAGE과 PROTEOMAGE, 네덜란드 이​​니셔티브 유전체학에 의한 노화 (NFN S93)에서 국립 연구 네트워크 / 학술 연구에 대한 네덜란드기구 (NGI / NWO, 05040202과 050 - 060-810 NCHA), 유럽 연합 (EU)은 우수 수명 (FP6 036894)의 네트워크를 재정 지원하고, 유전체학에 대한 혁신 지향 연구 프로그램 (SenterNovem, IGE01014 및 IGE5007).

1. 플라스미드 건설 <ol><li> 주문 맞춤 oligo-세포핵의 dgn (ACKNWFSSLSHFVIHL 11) 및 dgnFS (HARTGSLACPTSSSICE)에 대한 인코딩 및 dgn / dgnFS (그림 1)과 함께 GFP의 융합을 얻기 위해 pEGFP-C1 벡터로를 ligate. </li><li> pENTR 방향 TOPO의 복제 키트의 프로토콜과 pLenti6/V5 방향 TOPO 복제 키트 (그림 6)를 계속에 따라 PCR에 의해 GFP-dgn과 GFP-dgnFS의 코딩 순서를 증폭. </li></ol> 2. 바이러스 제작 <ol><li> 그들은 transfection의 날에 90~95%의 합류 될 수 있도록 T75 플라스크에 HEK 293FT 세포에서 transfection 전날 (1 일) 씨. </li><li> transfection의 날 혈청과 15 ML 매의 항생제없이 DMEM의 1.5 ML에 lipofectamine 2,000 시약 36 μl를 희석. 또 다른 15 ML 매를 사용하여 두 GFP에 대한 보완 DNA가 구축 들고 3 μg pLenti6/V5을 희석<html> - dgn 또는 GFP-dgnFS, 혈청과 항생제가없는 DMEM의 1.5 ML의 2.5 μg 봉투 인코딩 플라스미드 pMD2.G와 7.5 μg 벡터 백본 psPAX2. 5 분 후에 희석 된 DNA가 희석 lipofectamine 2,000 시약과 결합되어 있습니다. </li><li> 양식에 DNA-lipofectamine 단지를 허용하도록 실온에서 20 분의 혼합물을 품다. </li><li> HEK 293FT 세포의 매체를 제거 중 7 ML (항생제 제외)으로 조심스럽게 교체하고 혼합에 대한 술병과 앞뒤로 바위를 (2 일)에 DNA-lipofectamine 혼합물을 추가합니다. humidified 5% CO 2 인큐베이터에서 37 ° C에서 밤새 술병을 품다. </li><li> 중간 다음 날 (; 3 일 항생제없이 10 ML 매체) 변경합니다. </li><li> 48 시간 후 (5 일) 수확 표면에 뜨는, 룸 온도에서 5 분 300 XG에서 원심 분리기와 0.45 μm PVDF 필터를 통해 표면에 뜨는을 필터링합니다. </li></ol> 문화 매체 - DMEM (HEK 293FT) 십t &quot;&gt; DMEM 10% FBS 0.1 MM 가상 NEAA 6 MM L-글루타민 1 ㎜의 가상 나트륨 Pyruvate 1퍼센트 펜 - 패 혈성 (선택 사항) 500 μg / ML Geneticin (선택 사항) 3. 폴리에틸렌 글리콜 (PEG) 강수량의 바이러스 농도 <ol><li> 4의 표면에 뜨는과 2 시간에 품다 ° C. 네 개의 볼륨, 하나의 볼륨 폴리에틸렌 글리콜 솔루션을 (PEG 50 ㎜의 폴리에틸렌 글리콜, 41 MM NaCl, 압력솥, 산도 = 7.2)를 추가 조심스럽게 그것을 반전으로 모든 20-30 분 섞는다. </li><li> 4 ° C.에 30 분에 1,500 XG에 스핀 다운 흰색 펠렛이 표시됩니다. </li><li> 표면에 뜨는을 대기음, 4 ° C.에서 5 분 1500 XG에서 다시 원심 분리기 나머지 PEG 솔루션을 기음. </li><li> 중간하거나 펠렛을 Resuspend 인산염 버퍼 생리 (PBS)를 pipetting 아래에서 20 명 정도로 30 초 힘차게 소용돌이에 의해. 가이드 라인은 한 T75 술병과 나누어지는 그 100 μl에 500 μl를 사용하여 있습니다. -80에서 바이러스를 저장 ° C. </li></ol><ol><li> 5 아웃 씨 × 6 잘 판 transfection 전날의 각 잘에 10 4 U2-OS 세포. </li><li> transfection의 날 문화 매체를 제거하고 DMEM 8 μg / ML polybrene (hexadimethrine 브롬화)를 포함하는 1 ML하여 교체하십시오. 농축 바이러스에게 표면에 뜨는 1시 10분과 1:100을 희석 잘 하나에 각각 1 μl를 추가합니다. 높은 바이러스 농도는 1 μl, 5 μl하고 나머지 우물에 집중 바이러스 표면에 뜨는 10 μl를 사용합니다. 하나는 잘 긍정적 인 제어로 남아 있습니다. </li><li> 다음 날 DMEM 2 ML하여 매체를 교체하십시오. </li><li> 선택 다음날로 시작합니다. 각도에 10 μg / ML Blasticidin을 적용합니다. 매초마다 하루 항생제 포함하는 매체를 교체하십시오. </li><li> 약 6-7일 선택을 시작한 후 untransduced 세포가 죽었어요. 착색은 PBS로 세포를 세 번 씻고 크리스탈 바이올렛 (10 MG / L 크리스탈 바이올렛과 ​​세포를 표지20 % 에탄올)과 상온에서 5-10 분에 품다 인치 기음 크리스탈 바이올렛은 (몇 번 재사용 할 수)와 ddH 2 O와 두 번 우물을 씻어 판을 건조. </li><li> 식민지을 계산하고, 1,000 해당 희석 (그림 7)에 곱합니다. 이 절차는 바이러스 / ML의 transfection 단위 (TU)를 계산 할 수 있습니다. </li></ol> 문화 매체 - DMEM (HFF-2/U2-OS) DMEM 10% FBS 6mM L-글루타민 1퍼센트 펜 - 패 혈성 5. 인간 Diploid의 섬유 아세포의 도입 (이 절차는 모든 세포 유형에 대해 사용할 수 있습니다.) <ol><li> 5 아웃 씨 × 10 4 인간의 diploid의 섬유 아세포 (HDFs) 6 - 잘 접시에 전달 전날. </li><li> 한 밀리리터의 총 8 μg / 전달 증강 등의 ML의 polybrene과 함께 두 감염의 다중성을 사용합니다. </li><li> 다음 날 중간 변경합니다. </li><li> 언제세포가 confluency 시작 10 μg / ML blasticidin로 선택의 70~80% 이용하실 수 있습니다. 선택이 완료되면 (더 이상 untransfected 세포가 죽는 없음) 세포의 확장이 시작 수 있으며, 세포 실험을위한 준비가되어 있습니다. blasticidin과 만성 선택은 안정적인 세포 라인 overexpressing GFP-dgn 또는 GFP-dgnFS 융합 단백질, proteasome 활동을 측정 할 준비를 생성 할 수 있습니다. 이 절차는 셀 타입의 독립적 인 매우 높은 transfection 효율을 보장합니다. </li></ol> 6. 유동 세포 계측법에 의한 측정 <ol><li> 1 아웃 씨 × 10 5 세포 (GFP-dgn 또는 GFP-dgnFS 중 하나를 들고) 실험 전날. </li><li> 제어 세포에게 사전에 3 시간 proteasome 억제제와 측정, 예를 들어 100 μg / ML의 LLnL을 취급합니다. </li><li> PBS로 두 번 세척하고 트​​립신의 0.5 ML를 사용하여 세포를 수확. trypsinisation 문화 매체의 4.5 ML을 사용하고 15 ML 팔콘로 셀을 전송 중지합니다. </li><li> 스핀 t상온에서 5 분 300 XG에서 그는 세포. </li><li> 37, 매체를 대기음 PBS에있는 세포를 resuspend 5 분 300 XG에서 다시 스핀 다운 ° C. </li><li> FACS 버퍼 (10 MM의 Hepes, 140 MM NaCl, 2.5 MM CaCl 2, 산도 = 7.4) 400 μl에 resuspend PBS를 대기음 및 FACS 튜브에 세포를 전송합니다. </li><li> 유동 세포 계측법에 의해 얼음과 측정 샘플에있는 세포를 유지. 셀은 4 ° C.에 이상 30 분에 저장하지 않아야합니다 </li></ol> 7. 대표 결과 GFP-dgn 융합 단백질은 proteasome 따라서 단백질이 즉시 타락을 타겟으로하는 시퀀스를 수행, 그것은 GFP 형광 신호의 감소에 해당합니다. frameshift 돌연변이 (GFP-dgnFS)이 순서의 변형 버전을 들고 proteasome에 의해 저하가 아니라 높은 녹색 형광로 연결됩니다. 이러한 이유로, 젊은 예상 높은 proteasome 활동으로 HDFs하고 GFP-DG와 transducedN 유동 세포 계측법 측정 모두와 epifluorescence (그림 2A와 B, 그림 3)의 낮은 (6 % 긍정적 인 세포) 형광 신호를 보여줍니다. 같은 HDFs 긍정적 인 세포의 39.7 %를 표시 GFP-dgnFS와 transduced. proteasome 억제제를 LLnL (N-아세틸-L-leucyl-L-leucyl-L-norleucinal)과 세포의 치료, GFP-dgn과 GFP-dgnFS 세포 (그림 2A와 B 모두에서 62.9 %의 최대 신호를 제기 ). 고령자 인간의 피부 샘플에 proteasome 활동에 가능한 감소를 확인하려면, 젊은 중년 및 이전 기증자로부터 격리 피부 섬유 아세포는 GFP-dgn과 GFP-dgnFS 감염된 위의 설명 및 유동에 의한 분석을하기 전에 동일한 통로 번호로 재배 세포 계측법. 이 실험에서, 젊은 사람들 사이 GFP 신호의 분명한 증가 (11.2 ± 0.88 % GFP 양성 세포)와 중년이 기증자는 (20.4 ± 2.27 % GFP 양성 세포, P = 0.003)을 나타내는, 관찰되었습니다 프로 티어 식물 감소노인 기증자 7 (그림 4)에서 얻은 샘플에 ome 활동. proteasome 활동에 더 감소는 오래된 개인 (그림 4)에서 분리 섬유 아세포에서 관찰되지 않았습니다. GFP-dgnFS에 대한 형광 강도는 모든 경우에 있었어요 ~ 90 % (데이터가 게재되지 않음) 세 가지 다른 연령 그룹에 대한 높은 transfection 효율을 나타냅니다된다. <img src="/files/ftp_upload/3327/3327fig1.jpg" alt="그림 1" /> 1 그림. GFP-dgn과 GFP-dgnFS 순서. 표시는 GFP (녹색) pEGFP-CL1 벡터 (회색)과 dgn / dgnFS 순서 (빨간색)의 여러 복제 사이트의 3&#39; 엔드입니다. <img src="/files/ftp_upload/3327/3327fig2.jpg" alt="그림 2" /> 그림 2. GFP-dgn과 GFP-dgnFS 인간의 diploid의 섬유 아세포의 유동 세포 계측법 분석. A. 젊은 사람의 포피의 섬유 아세포 (HFF-2)은 녹색 형광 단백질을 운반 lentiviral 벡터에 감염되었다 (GFP) dgn 유전자 또는 GFP-dgnFS (frameshift)는 등의 표시 및 유동 세포 계측법을 (FACS 캔토 II, Becton 디킨슨)를 사용하여 GFP 형광에 대한 분석 구성합니다. 표시 할 경우, 세포는 proteasome 억제제 N-아세틸-L-leucyl-L-leucyl-L-norleucinal (LLnL)로 3h 치료를했다. 감염되지 않은 세포는 제어로 사용되었습니다. 실험은 triplicates에서 수행되었다. B. 데이터 세 독립적 인 실험을 대표합니다. 번호는 GFP 양성 세포의 양을 반영합니다. <a href="http://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/3327/3327fig3.jpg" alt="그림 3" /> 그림 3. GFP-dgn과 GFP-dgnFS HFF-2 세포의 형광 현미경 분석. HFF-2 영은 그림 2에서와 같이 취급되었다. 감염 후 구일에서 셀은 V 있었다형광과 위상 대비 현미경으로 isualized. <img src="/files/ftp_upload/3327/3327fig4.jpg" alt="그림 4" /> 4 그림. 인간의 피부 노화에 proteasome 활동의 변화. 지정된 연령 그룹의 9 개 다른 기증자로부터 인간의 포피의 섬유 아세포는 최소한 확장과 GFP-dgn에 대한 코딩 lentiviral 구조에 감염되었습니다. 감염 후 구일에서, 셀 유동 세포 계측법에 의해 분석 하였다. 데이터 중복의 연령 그룹 당 3 샘플 (± SE)에 취득했다. <img src="/files/ftp_upload/3327/3327fig5.jpg" alt="그림 5" /> 그림 5. 실험 방법은. dgn와 dgnFS에 대한 사용자 정의 - oligo 세포핵은 pEGFP-C1 벡터로 복제되어 바이러스는 HEK 293FT 세포를 사용하여 각 구조에 대한 생산됩니다. 바이러스의 titer가 결정된다. 세포는 바이러스에 transduced 및 확장하고 있습니다. 선호 치료 후 세포 분석유동 세포 계측법에 의한 형광 신호. <img src="/files/ftp_upload/3327/3327fig6.jpg" alt="그림 6" /> 6 그림. pLenti GFP-dgn의지도는. GFP-dgn 시퀀스를 포함하여 pLenti6/V5-DEST 벡터의지도가 표시됩니다. 약어 : PCMV (CMV 프로모터), GFP-dgn (GFP-dgn의 순서), P SV40 (SV40 초기 발기인), EM7 (EM7 발기인), Blasticidin (Blasticidin 저항 유전자), ΔU3 / 3&#39;LTR (3&#39;LTR로 삭제 U3 지역), SV40 PA (SV40 polyadenylation 신호), 암피실린 (암피실린 저항 유전자), pUC 오라 (pUC 원본), PRSV / 5&#39;LTR (RSV / 5&#39;LTR 하이브리드 발기인), Ψ (HIV-1 Ψ 포장 신호 ), RRE (HIV-1 수익 응답 요소). <img src="/files/ftp_upload/3327/3327fig7.jpg" alt="그림 7" /> 그림 7. U2-OS의 titering 판. U2-OS는 6 잘 판에 나가 놓는하고 다른 바이러스 농도 (1백분의 1과 1 / 10의 희석, 1, 5 및 공동 10 μl와 transfected되었습니다) 바이러스 표면에 뜨는을 ncentrated. 하나는 잘 untransfected 제어 (NT)로 사용되었다. 6-7일 후, 전지 크리스탈 바이올렛, 공기 건조 물들했다.

<ol>
	<li>Coux, O., Tanaka, K., Goldberg, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+20S+and+26S+proteasomes.">Structure and functions of the 20S and 26S proteasomes.</a> Annu. Rev. Biochem. 65, 801-847 (1996).</li><li>Davies, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+oxidized+proteins+by+the+20S+proteasome.">Degradation of oxidized proteins by the 20S proteasome.</a> Biochimi. 83, 301-310 (2001).</li><li>Stangl, K., Stangl, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+pathway+and+endothelial+(dys)function.">The ubiquitin-proteasome pathway and endothelial (dys)function.</a> Cardiovasc. Res. 85, 281-290 (2009).</li><li>Tuoc, T. C., Stoykova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+ubiquitin-proteosome+system+in+neurogenesis.">Roles of the ubiquitin-proteosome system in neurogenesis.</a> Cell Cycle. 9, 3174-3180 (2010).</li><li>Rock, K. L., 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=Inhibitors+of+the+proteasome+block+the+degradation+of+most+cell+proteins+and+the+generation+of+peptides+presented+on+MHC+class+I+molecules.">Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.</a> Cell. 78, 761-771 (1994).</li><li>Lehman, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+proteasome+system+in+neuropathology.">The ubiquitin proteasome system in neuropathology.</a> Acta Neuropathol. 118, 329-347 (2009).</li><li>Koziel, R., Greussing, R., Maier, A. B., Declercq, L., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Interplay+between+mitochondrial+and+proteasome+activity+in+skin+aging.">Functional Interplay between mitochondrial and proteasome activity in skin aging.</a> J. Invest. Dermatol. 131, 594-603 (2010).</li><li>Grillari, J., Grillari-Voglauer, R., Jansen-Durr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+cellular+proteins+by+ubiquitin+and+ubiquitin-like+molecules:+role+in+cellular+senescence+and+aging.">Post-translational modification of cellular proteins by ubiquitin and ubiquitin-like molecules: role in cellular senescence and aging.</a> Adv. Exp. Med. Biol. 694, 172-196 (2010).</li><li>Bulteau, A. L., Szweda, L. I., Friguet, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+declines+in+proteasome+activity+in+the+heart.">Age-dependent declines in proteasome activity in the heart.</a> Arch. Biochem. Biophys. 397, 298-304 (2002).</li><li>Strucksberg, K. H., Tangavelou, K., Schroder, R., Clemen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasomal+activity+in+skeletal+muscle:+A+matter+of+assay+design,+muscle+type,+and+age.">Proteasomal activity in skeletal muscle: A matter of assay design, muscle type, and age.</a> Anal. Biochem. , (2009).</li><li>Bence, N. F., Sampat, R. M., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+the+ubiquitin-proteasome+system+by+protein+aggregation.">Impairment of the ubiquitin-proteasome system by protein aggregation.</a> Science. 292, 1552-1555 (2001).</li><li>Dantuma, N. P., Lindsten, K., Glas, R., Jellne, M., Masucci, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-lived+green+fluorescent+proteins+for+quantifying+ubiquitin/proteasome-dependent+proteolysis+in+living+cells.">Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells.</a> Nat. Biotechnol. 18, 538-543 (2000).</li><li>Lindsten, K., Menendez-Benito, V., Masucci, M. G., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+mouse+model+of+the+ubiquitin/proteasome+system.">A transgenic mouse model of the ubiquitin/proteasome system.</a> Nat. Biotechnol. 21, 897-902 (2003).</li><li>Liu, J., 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=Impairment+of+the+ubiquitin-proteasome+system+in+desminopathy+mouse+hearts.">Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.</a> FASEB J. 20, 362-364 (2006).</li><li>Bowman, A. B., Yoo, S. Y., Dantuma, N. P., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+dysfunction+in+a+polyglutamine+disease+model+occurs+in+the+absence+of+ubiquitin-proteasome+system+impairment+and+inversely+correlates+with+the+degree+of+nuclear+inclusion+formation.">Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation.</a> Hum. Mol. Genet. 14, 679-691 (2005).</li><li>Myung, J., Kim, K. B., Lindsten, K., Dantuma, N. P., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+proteasome+active+site+allostery+as+revealed+by+subunit-specific+inhibitors.">Lack of proteasome active site allostery as revealed by subunit-specific inhibitors.</a> Mol. Cell. 7, 411-420 (2001).</li><li>Menendez-Benito, V., Heessen, S., Dantuma, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+ubiquitin-dependent+proteolysis+with+green+fluorescent+protein+substrates.">Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.</a> Methods Enzymol. 399, 490-511 (2005).</li><li>Lener, B., 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=The+NADPH+oxidase+Nox4+restricts+the+replicative+lifespan+of+human+endothelial+cells.">The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells.</a> Biochem. J. 423, 363-374 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

ubiquitin proteasome - 활동 기자 기판으로 녹색 형광 단백질 (GFP)을 사용하여 첫 번째 출판 2000 12 년에 출판되었다. 그 후, GFP 세포 활동, 특히 ubiquitin-proteasome 프로세스를 시각화 할 수있는 일반적인 도구가되었습니다. 생체 내 ubiquitin-proteasome 활동을 모니터링 할 수 GFP 기반 기자와 유전자 변형 마우스 모델은 13 도입되었습니다. 생체 연구에 추가이 간행물 14에서 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> pEGFP-C1 벡터 </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR 방향 TOPO 복제 키트 </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 방향 TOPO 복제 키트 </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 시약 </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> 시그마 </td><td> D5546 </td></tr><tr><td> PVDF 필터 (Rotilabo-Spritzenfilter) </td><td> 로스 </td><td> P667.1 </td></tr><tr><td> 폴리에틸렌 글리콜 </td><td> 시그마 </td><td> P2139 </td></tr><tr><td> NaCl </td><td> 머크 </td><td> 1.06404.1000 </td></tr><tr><td> Dulbecco의 인산이 Sali 버퍼NE 1X (PBS) </td><td> Invitrogen </td><td> 14,190 </td></tr><tr><td> hexadimethrine 브로마이드 </td><td> 시그마 </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> 크리스탈 바이올렛 </td><td> 시그마 </td><td> C3886 </td></tr><tr><td> FACS 튜브 </td><td> BD Biosciences </td><td></td></tr><tr><td> 페니실린의 스트렙토 마이신 (펜 - 패 혈성) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-글루타민 200 MM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> 가상 비 필수 아미노산 (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> 가상 나트륨 Pyruvate 100 MM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-포도당 (45 %) </td><td> 시그마 </td><td> G8769 </td></tr><tr><td> Geneticin </td><td> Invitrogen </td><td> 11811023 </td></tr><tr><td> CaCl2 </td><td> 머크 </td><td> C5080 </td></tr><tr><td> Hepes </td><td> 시그마 </td><td> H3375 </td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) (0.05 %) </td><td> Invitrogen </td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteasome은 이상, misfolded, 손상되었거나 산화 단백질 1, 2의 proteolytic 저하에 관련된 주요 세포 세포 기관입니다. proteasome 활동의 유지 보수는 세포의 스트레스 반응 3, 세포주기 조절과 세포 분화 4 면역 체계의 반응 5와 같은 많은 주요 세포 프로세스에 연루되었습니다. ubiquitin proteasome - 시스템의 장애는 종양 및 neurodegenerative 질병 4, 6의 개발에 관한되었습니다. 또한, proteasome 활동에 감소는 세포 노화 및 노화 organismal 7, 8, 9, 10의 기능으로 발견되었다. 여기, 우리는 GFP-dgn 융합 단백질을 사용하여 생활 세포에 ubiquitin-proteasome 활동을 측정하는 방법을 제시한다. 기본 세포를 생활에 ubiquitin-proteasome 활동을 모니터링 할 수 있도록, 보완 DNA는 (녹색 형광 단백질 (GFP) dgn 융합 단백질에 대한 코딩 구성합니다 GFP-dgn, 불안정)과 frameshift 변이 (GFP-dgnFS, 안정적 11) 운반 변종 lentiviral 표현 벡터에 삽입되어 있습니다. 이 셀 형식이나 기증자의 나이와 무관 한 매우 높은 transfection 효율을 보장하기 때문에 우리는 기존의 transfection 기술을 통해이 기술을 선호합니다. GFP-dgnFS (안정적) 단백질과 proteasome 억제제의 부재 또는 존재의 불안정 단백질 (GFP-dgn)에 의해 표시되는 형광의 차이점은 각 특정 세포 변형에 ubiquitin-proteasome 활동을 추정하는 데 사용할 수 있습니다. 이러한 차이는 epifluorescence 현미경에 의해 모니터링 될 수 있거나 유동 세포 계측법에 의해 측정 할 수 있습니다.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

생활 세포의 ubiquitin proteasome - 활동을 모니터링하는 방법이 설명되어 있습니다. degron-불안정 GFP-(GFP-dgn)과 안정적인 GFP-dgnFS 융합 단백질이 생성되고 lentiviral 발현 벡터를 사용하여 셀에 transduced 있습니다. 이 기술은 ubiquitin-proteasome 활동이 쉽게 epifluorescence 또는 유동 세포 계측법을 사용하여 평가 할 수있는 안정적인 GFP-dgn/GFP-dgnFS 표현 세포 라인을 생성 할 수 있습니다.

살아있는 세포에서 Ubiquitin-proteasome 활동 모니터링 Degron (dgn) - 불안정 그린 형광 단백질 (GFP) 기반 리포터 단백질을 사용하여

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

Research

JoVE Journal

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

16.6K 조회수.  Institute for Biomedical Aging Research. 생활 세포의 ubiquitin proteasome - 활동을 모니터링하는 방법이 설명되어 있습니다. degron-불안정 GFP-(GFP-dgn)과 안정적인 GFP-dgnFS 융합 단백질이 생성되고 lentiviral 발현 벡터를 사용하여 셀에 transduced 있습니다. 이 기술은 ubiquitin-proteasome 활동이 쉽게 epifluorescence 또는 유동 세포 계측법을 사용하여 평가 할 수있는 안정적인 GFP-dgn/GFP-dgnFS 표현 세포 라인을 생성 할 수 ?...

비디오: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

배아 줄기 세포 생활에서 염색질 단백질 역학을 측정하는 Assays (FRAP 및 플립)를 Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

단백질 분해의 체계적인 분석을위한 기자 기반의 성장 분석

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

살아있는 세포의 단백질 조각 보완 분석 (PCA)에 의해 게놈 전체 단백질 - 단백질 상호 작용 상영

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

높은 처리량 시퀀싱을 활용하여 전체 - 단백질 포화 돌연변이 유발 도서관의 기능 평가를위한 프로토콜

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...