Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

<ol>
	<li>Pugh, R. 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=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., 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=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., 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=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. M., 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=Topology+based+identification+and+comprehensive+classification+of+four-transmembrane+helix+containing+proteins+(4TMs)+in+the+human+genome.">Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome.</a> BMC genomics. 17, 268 (2016).</li><li>Zou, Z., 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=Genome-Wide+Identification+of+Jatropha+curcas+Aquaporin+Genes+and+the+Comparative+Analysis+Provides+Insights+into+the+Gene+Family+Expansion+and+Evolution+in+Hevea+brasiliensis.">Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis.</a> Front Plant Sci. 7, 395 (2016).</li><li>Gilotti, A. C., 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=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. 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=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. 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=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., 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=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, S., 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=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

저자는 공개 아무것도 없어.

여기에보고 된 프로토콜은 혈관 세포 1 헤파린 수용체로서 TMEM184A의 식별 확증 증거를 제공하도록 설계되었다. 최저 기술은 정기적으로 신규 단백질의 식별을 확인하기 위해 하나의기구로 사용된다. 그러나, 최저 후 일부 기능적 손실 후보 단백질 실제로 정확한 수용체 충분한 증거 (또는 다른 기능성 단백질)은 일반적 아니다. 이는 후보 단백질이 실제로 기능을 발휘한다는 증?...

새로운 기능에 대한 후보 단백질의 식별은 종종 부분 서열 결정 하였다 선호도 기반 격리 프로토콜에 따라 달라집니다. 새로 확인 된 단백질의 최근 사례는 횡단 단백질 184A (TMEM184A), 헤파린 친 화성 상호 작용 1 일 이후에 확인 된 헤파린 수용체 및 TgPH1, 포스의 PI (3,5) P (2) (2)를 결합하는 플렉스 트린 상동 도메인 단백질을 포함한다. 다른 신규 한 단백질의 식별은, 등 비타 의한 것과 펩티드의 직접적인 서열 분석을 포함한다. 누가 미리지지 않은 유전자에서 3 단백질 제품을 식별하는 트랜스 펩티드를 사용했다. 이와 유사하게, 신규 한 단백질 서열의 확인은 새로운 4TM 단백질 (4)의 식별 앞서있어서 단백질 패밀리의 검색 생물 정보학을 이용하여 달성 될 수있다. 아쿠아 포린 가족 유전자 서열의 검사 등이있다그래서 새로운 기능 5 새로운 회원의 식별을 얻었다. 식별 한 후, 단백질의 기능 분석은 종종 예컨대 아쿠아 포린 마찬가지로 단백질 기능에 대한 구체적인 분석을 이용하여 조사 할 수있는 다음 단계는 전형적이다. 가능하면, 새로 확인 된 단백질의 기능은 특정 효소 또는 유사한 생체 외 기능 분석으로 관찰 할 수있다. 새로운 단백질의 많은 기능은 체외 분석에서 만 그대로 세포 또는 유기체에서 발생하는 복잡한 상호 작용에 의존하기 때문에 항상 적용되지 않습니다. 그러나, 생체 내 분석은 그들이 유전자 서열에 의존하는 방식으로 설계되어야한다. 세포 배양 및 / 또는 간단한 모델 생물에서 최저 단백질 / 기능 식별 6 뒷받침하는 근거를 제공 할 수 있습니다. 전술 한 바와 같이 식별 된 신규 단백질,이 기능을 확인하기 위해 단순히 단백질 노크 종종 불충분D 유전자 서열에 의존 생체 기능 분석의 디자인은 신규 한 단백질의 특성에 중요해진다. 최저의 식별과 일치하는 결과를 얻었다 후 헤파린 수용체로서 TMEM184A 최근 식별은 친 화성 크로마토 그래피와 MALDI MS 1을 사용하는 (즉, 혈관 평활근 및 내피 세포에서 염증 반응에서 확산 변조), 7은 분석의 컬렉션을 개발할 수있는 기회를 제공 . 최근의 검토 헤파린은 다양한 성장 인자, 이들의 수용체, 세포 외 매트릭스 성분, 세포 부착 수용체와 다른 단백질 8 구체적으로 작용 함을 확인 하였다. 혈관계, 헤파린 및 헤파 란 설페이트 프로테오글리칸에 수백 9 단백질과 상호 작용 (헤파린 유사한 구조 헤파 란 황산 사슬 함유). 기능적으로 그쪽을 확인하려면t TMEM184A는 헤파린 흡수 및 결합에 관여하고, TMEM184A의 유전자 구조를 이용 기술이 개발되었다. 본 보고서는 GFP-TMEM184A에 기초 분석법 모음 헤파린 수용체 TMEM184A 등의 신원 확인에 사용하기 위해 구성 포함한다.

<table><tbody><tr><td>GFP-TMEM184A construct</td><td>OriGene</td><td>RG213192</td><td></td></tr><tr><td>Rhodamine-Heparin</td><td>Creative PEGWorks</td><td>HP-204</td><td>Light Sensitive</td></tr><tr><td>Fluorescein-Heparin</td><td>Creative PEGWorks</td><td>HP-201</td><td>Light Sensitive</td></tr><tr><td>Mowiol</td><td>EMD Millipore</td><td>475904-100GM</td><td></td></tr><tr><td>Paraformaldehyde (PFA, methanol free)</td><td>Thermo Sci Pierce Biotech, available through Fisher Scientific</td><td>PI28908 at Fisher</td><td>Use in Fume Hood</td></tr><tr><td>Reacti-bind neutravidin plates (Avidin coated black 96-well dishes)</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>PI15510 at Fisher</td><td>Pay attention to shelf-life</td></tr><tr><td>Black 96-well plates</td><td>Corning Life Sciences Plastic, purchased through Fisher Scientific</td><td>064432 at Fisher</td><td></td></tr><tr><td>A7r5 vascular smooth muscle cell line</td><td>ATCC</td><td>CRL 1444</td><td>Can be exchanged into MEM medium&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>BAOEC bovine aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>B304-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1,7&lt;/sup&gt;</td></tr><tr><td>BAOSMC bovine aortic smooth muscle cells</td><td>Cell Applications, Inc.</td><td>B354-05</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;1&lt;/sup&gt;</td></tr><tr><td>RAOEC rat aortic endothelial cells</td><td>Cell Applications, Inc.</td><td>R304-05a</td><td>Culture as recommended initially, can be exchanged into MEM medium for continuing culture&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>Biotinylated anti-GFP</td><td>Thermo Sci Pierce Biotech, through Fisher Scientific</td><td>MA5-15256-BTIN</td><td></td></tr><tr><td>Streptavidin-coated beads</td><td>Sigma</td><td>S1638</td><td></td></tr><tr><td>HeBS</td><td>Available from Bio-Rad</td><td></td><td>Can be prepared in the lab. The pH is 6.8</td></tr><tr><td>TMEM184A antibody to the N-terminus</td><td>Santa Cruz Biotechnology</td><td>sc292006</td><td>Only known TMEM184A antibody to N-terminal region.</td></tr><tr><td>TMEM184A antibody to the C-terminus</td><td>Obtained from ProSci Inc, Poway, CA&amp;nbsp;</td><td>Pro Sci 5681</td><td>ProSci used in &lt;strong&gt;Figure 1&lt;/strong&gt;</td></tr><tr><td>GFP antibodies</td><td>Santa Cruz Biotechnology</td><td>sc9996</td><td>Used in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Secondary antibodies, labeled with TRITC or Cy3</td><td>Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td><td>711 025 152 (donkey anti-rabbit, TRITC)&lt;br/&gt; 715 165 150 (donkey anti-mouse, Cy3)</td><td>Minimal cross-reactivity to minimize any nonspecific staining.</td></tr><tr><td>CHAPS</td><td>Purchased from Sigma</td><td>C5849</td><td>Note that this specific catalog number has been discontinued.&amp;nbsp; Supplier will provide information regarding replacement.</td></tr><tr><td>Live imaging 35-mm dishes</td><td>MatTek (Ashland MA)</td><td>P35G-1.0 &amp;ndash; 20 mm - C</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 510 Meta with a 63X oil-immersion lens</td><td>Used for images and live-imaging in &lt;strong&gt;Figures 1, 2 and 3&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>C2+ confocal with a 60X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>Zeiss LSM 880 with a 63X oil-immersion lens</td><td>Used for images in &lt;strong&gt;Figure 2C&lt;/strong&gt;</td></tr><tr><td>Electroporation equipment</td><td>Bio-Rad</td><td>Gene Pulser X-Cell System</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Available from MidSci</td><td>EC2L</td><td>Can also be obtained from equipment supplier</td></tr><tr><td>Plate reader</td><td>TECAN</td><td>TECAN Infinite&amp;reg; m200 Pro plate reader</td><td>Readings in the middle of the wells rather than at the surface.</td></tr><tr><td>Computer program for measuring staining intensity</td><td>Image J</td><td>https://imagej.nih.gov/ij/&lt;br/&gt; Program and information available on-line</td><td>Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Cell Culture trypsin solution</td><td>Sigma</td><td>T4174</td><td>purchased as a 10x solution</td></tr></tbody></table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

새로운 단백질 선호도 기반 격리 및 생물 정보학 분석을 통해 확인하면, 그들은 종종 크게지지 않은 있습니다. 예측 된 시퀀스 내의 특정 펩타이드에 대한 항체는 일부 현지화 실험을 할 수 있습니다. 그러나, 다른 항체와의 상호 작용에 종종 배제 할 수 없다. 이러한 상황은 단백질 서열 분석에 의존 세트를 개발하기위한 기회를 제공했다. 특히, 단백질의 C 말단에 GFP 코딩 서열에 연결된 유전자 서열을 함유하는 작 제물을 수득하고 이러한 목적으로 사용 하였다. 실험 지역화 리간드 친 화성을 특징, 기능 및 이득은 원래 설계 헤파린 수용체 1과 TMEM184A의 식별을 확인하기 위해 수행되었다. 또한, 막 구조는 토폴로지 질문 상세한 단백질 - 리간드 상호 작용을 어드레싱 연구에 사용될 수있다. 본 보고서는 아칸소를 제공합니다쉽게 다른 새로운 단백질을 위해 적용 할 수있는 혈관 세포에서 발현되는 GFP-TMEM184A 구조를 기반으로 실험 프로토콜의 플랜지.

이론적으로, 어떤 DNA의 형질 전환은 친 유성 형질 전환 시약으로 수행 할 수있는 세포로 구성,하지만, 이전 보고서는 GFP의보다 효과적인 형질은 전기 (12)를 사용하여 내피 세포로 구성한다 나타냅니다. 여기서 제공되는 프로토콜은 통상적으로 사용되는 주 유래 내피 세포 및 평활근 세포의 80 % 이상에서 발현은 GFP-구성하였습니다. 고용 구조의 디자인은 ...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

진핵 세포 발현을 위해 디자인 된 카르복시 말단에 GFP 태그 컨스 부호화 TMEM184A는 혈관 세포에서 헤파린 수용체로서 TMEM184A의 식별을 확인하기위한 분석에 사용 하였다.

GFP - 태그 TMEM184A를 사용하면 헤파린 수용체 신원 확인을 위해 구축

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

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JoVE Journal

Genetics

7.9K 조회수.  Lehigh University.  진핵 세포 발현을 위해 디자인 된 카르복시 말단에 GFP 태그 컨스 부호화 TMEM184A는 혈관 세포에서 헤파린 수용체로서 TMEM184A의 식별을 확인하기위한 분석에 사용 하였다. 

비디오: Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

식 및 단일 입자 Cryo 전자 현미경 검사 법에 의하여 구조 결심에 대 한 인간의 지질 민감한 양이온 채널 TRPC3의 정화

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

연구

생화학

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

사진-가교 매핑 및 라이브 포유류 세포에서 Bioorthogonal 화학 GPCRs에 프로브 화학의 유전 결합을 최적화

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

화학

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

TCR-pMHC 바인딩 측정<em&gt; 현장에서</em&gt; FRET 기반의 현미경 분석을 사용하여

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

생체공학

<meta charset="utf8"></head><body>CRISPR/Cas9 Knock-In을 사용한 제브라피시 유충의 형광 단백질 태깅

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>제브라피시에서 저발현 단백질을 시각화하기 위한 형광 라벨링

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

제브라피시에서 저발현 단백질을 시각화하기 위한 형광 라벨링

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

유전학

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

GABA 수용체 막 현지화 및 인신 매매를 연구하는 α-Bungarotoxin 바인딩 사이트 태그를 사용하여

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

신경과학

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

이미징 단백질 - 단백질 상호 작용 생체내에</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

생물학

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

유전자 인코딩 분자 프로브는 G 단백질 결합 수용체를 연구하는

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

유동 세포 계측법을 사용하여 재조합 이온 채널의 상대 셀 표면 및 전체 식의 결정

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

GFP - 태그 TMEM184A를 사용하면 헤파린 수용체 신원 확인을 위해 구축

요약

더 많은 비디오 탐색

이 비디오의 챕터

식 및 단일 입자 Cryo 전자 현미경 검사 법에 의하여 구조 결심에 대 한 인간의 지질 민감한 양이온 채널 TRPC3의 정화

사진-가교 매핑 및 라이브 포유류 세포에서 Bioorthogonal 화학 GPCRs에 프로브 화학의 유전 결합을 최적화

TCR-pMHC 바인딩 측정

제브라피시에서 저발현 단백질을 시각화하기 위한 형광 라벨링

제브라피시에서 저발현 단백질을 시각화하기 위한 형광 라벨링

제브라피시에서 저발현 단백질을 시각화하기 위한 형광 라벨링

GABA 수용체 막 현지화 및 인신 매매를 연구하는 α-Bungarotoxin 바인딩 사이트 태그를 사용하여

이미징 단백질 - 단백질 상호 작용 생체내에

유전자 인코딩 분자 프로브는 G 단백질 결합 수용체를 연구하는

유동 세포 계측법을 사용하여 재조합 이온 채널의 상대 셀 표면 및 전체 식의 결정