Biz, Ulusal Sağlık Enstitüleri (1R21CA138373 teşekkür ediyor ve bu çalışmanın finansal destek Kanseri (SU2C) Stand Up. HY, Amerikan Kanser Araştırma, Sidney Kimmel Vakfı Kimmel Scholar Ödülü Derneği, 2009 Elion Ödülü için minnettar Kanser Araştırma (SKF-08-101) ve Ulusal Bilim Vakfı Fakültesi Erken Kariyer Ödülü (NSF0954819).

1. Klonlama Hususlar <ol><li> Ticari olarak hazırlanan oligonükleotidler NheI ve BamHI kısıtlama siteleri ve 5&#39;-fosforile ile çevrili ilgi TMD temsil pTox7 (BamHI kısıtlama sitesi 14 doğrudan sonra tek bir baz çifti ekleme laboratuvarımızda güncellenmiştir) (Şekil 3) sırayla sindirilir içine bağlandı . BamHI ve NheI. Bir örnek oligonükleotid, aşağıda gösterilmiştir: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> TMD sıra 12-24 kalıntıları (kısa dizileri muhtemelen vektör kodlanmış hidrofobik artıkları uzun olacak) olmalıdır. Sıralı kalıntı eklemeler ve ToxR TMD göreli dönme birlikte kalıntı silmeler. 15,16 Nihayet, arabinoz konsantrasyonu 0.001 ve% 0.01 arasında değişmektedir olmalıdır arayüzü araştırmak için, dört değişik TMD tasarım oluşturulmalıdır (w / v) farklı TMD dizileri arasında β-galaktosidaz sinyalleri maksimum farklılıklar gözlenmektedir konsantrasyonunu belirlemek için farklı ekspresyon seviyeleri test koşulları altında farklı yakınlık en iyi ayırt edilebilir tanımlamak için tavsiye edilir. Arabinoz ve antibiyotiklere ek olarak, 0.4 mM IPTG farklı TMDS arasındaki yakınlık farkı artırmak için kullanılabilir. En az dört nüsha ToxR ölçümü yapılmalıdır. Tüm prosedürü farklı plazmid dönüşümler ile en az üç kez tekrar edilmelidir. 2. Bakteriyal Kültürler Büyüme <ol><li> Yavaşça 15 ml kültür tüp içine buz ve transfer FHK12 yetkili hücreleri (200 ul) Çözülme. Plazmid DNA (200 ng) ekleyin ve 30 dakika buz üzerinde hücreleri kuluçkaya yatmaktadır. </li><li> 42 90 s inkübasyon ısı-şok hücreleri ° C, 2 dakika süreyle buz üzerinde kuluçka takip. </li><li> SOC medya (800 ul) ekleyin ve 37 numunelerin inkübasyon ° C (300 rpm) sallayarak bir saat </li><li> Üç nüsha halinde 15 ml kültür tüpleri dönüşüm karışımı 50 ul kloramfenikol (30 mg / ml) ve arabinoz (0.0025% w / v) ile 5 ml LB medya inoküle edin. 37 numune ° C&#39;de 20 saat (300 rpm) sallayarak (5 ul kültür Alternatif 96-plaka orta 100 ul aşılamak için kullanılan olabilir, çok sayıda numune ile uğraşırken bu yöntem yararlıdır, hataları biraz daha yüksek olmasına rağmen, yol açacak buharlaşma önlemek için hata ile dış ortam kuyuları doldurup, ancak örnekler için onları kullanmayın. Sonunda), kapak ve parafilm ile plaka arasında ortak çift sarın. </li></ol> 3. Β-galaktosidaz Aktivite ölçümü <ol><li> Onceden plaka okuyucu ile 28 ° C </li><li> Üst katmanı (sulu) sadece almak için emin olun, büyük bir pipet ucu ile Z-tampon bir rezervuar içine aktarın. 96 plaka kuyulardan taze hazırlanmış Z-buffer/chloroform 100 ul aktarın. Her kültürün 5 ul dört nüsha plaka kuyuların içine aktarın. Dört kuyu boş olarak hizmet verecek kültür yok sayın. </li><li> Hücre yoğunluğu belirlemek için plaka OD 595 ölçün. </li><li> Plaka tüm kuyuları Z-buffer/SDS 50 ul ekleyin. Plaka plaka okuyucu hücreleri lyze için 10 dakika süreyle iyice çalkalayın. Hücre süspansiyonları lizis sonra açık ve gerekirse sallayarak adımı tekrarlayın emin olun. Eksik lizis Z-buffer/chloroform taze hazır değildi göstermektedir. </li><li> Tüm kuyulara Z-buffer/ONPG taze hazırlanmış 50 ul ekleyin ve 20 dakika boyunca her 30 s plaka okuyucu ve ölçü OD 405 plaka dönmek. </li><li> Aşağıdaki denklemi kullanarak (boş çıkarma hatırlamak) β-galaktosidaz aktivitesi hesaplayın. Doğrusal bir modeli uydurmak kullanarak 1,0 OD 405 aralığı 0.0 tüm veri noktalarını kullanarak, OD 405 / dk oranı hesaplanmalıdır . <img src="files/ftp_upload/2721/2721eq1.jpg" alt="Denklem 1" /> Miller birimleri, farklı günlerde bazen farklılık gösterir. Bu nedenle, genel not ortalaması gibi bir referans inşa, her bir test ölçülmelidir. Kendi değerlerini ToxR değerleri normalleşmesi için kullanılabilir. </li></ol> 4. Protein Ekspresyonu için kontrol <ol><li> Batı yapıları arasında bile protein ekspresyonu doğrulamak için blot gerçekleştirin. Bir mikrosantrifüj üç nüsha kültürler ve santrifüj 50 ul (2000 rpm, 4 dakika) birleştirin. Pipetleme süpernatantı ve 2 adet numune yükleme tamponu kalan pelet tekrar süspansiyon haline getirin. </li><li> % 8 standart bir jel üzerinde 7.5 ul Yük ve 1 saat 5 dakika boyunca 125 V electrophoreses yürütmek. Aktarımdan sonra, anti-MBP HRP-konjuge antikor ile inkübe ve görselleştirmek; kimerik protein yaklaşık 70 kDa, 48 kDa etrafında bazen görülen bazı yıkım ürünleri ile görülmektedir. Endojen MBP 45 kDa görülmektedir (bkz. Şekil 5). </li></ol>5. Uygun Membran Ekleme Kontrolü Maltoz bağlayıcı protein eksikliği hücre hattı kimerik TMD inşa uygun membran ekleme değerlendirmek için kullanılır. Tek karbon kaynağı olarak, maltoz ile minimal medya yetişen, sadece hücreleri periplasm doğru yer maltoz bağlayıcı protein ile bir zar-integral ifadesi ürün ifade büyümek mümkün. <ol><li> Transform PD28 hücreleri (FHK12 hücreleri için açıklandığı gibi) ve 2 ml LB orta aşılamak. 37 hücrelerin büyümesine ° C (300 rpm) bir gecede sallayarak. </li><li> Pelet 3500 rpm&#39;de santrifüj yoluyla hücreler, 10 dk, 4 ° C ve PBS (2 ml) tabanda büyük bir ucunda ya da yumuşak bir vorteks nazik pipetleme ile yıkayın. Pelet hücreleri (yukarıdaki gibi), ikinci kez PBS ile yıkayın pelet ve son olarak PBS (1 ml) tekrar süspansiyon haline getirin. </li><li> 37 az üç nüsha ve inkübe 5 ml minimal medya aşılamak için yeniden süspanse hücrelerinin 25 ul kullanın ° C sallayarak (300 rpm). 15-25 saat, plaka okuyucu kullanarak 96-plaka okuma ve her bir örnek 200 ul aktararak yaklaşık her 2 saat arasında OD 595 okuma alın. </li></ol> 6. Temsilcisi Sonuçlar: Şekil 4&#39;te gösterildiği transmembran etki alanı oligomerization eğilimi analiz ToxR transkripsiyonel muhabiri testinin kullanımı bir örnek. Önceleri biz oligomerization transmembran etki araştırıldı multispanning membran-integral protein latent membran protein-1 ToxR de dahil olmak üzere çeşitli teknikler (LMP-1) 14 Transmembran etki alanı beş (TM5) oligomerize güçlü bir eğilimi gösterirler gösterildi. Bu pozitif kontrol ile karşılaştırılabilecek yüksek Miller Üniteleri, GPA, köklü bir dimerizing dizisi olduğunu göstermiştir. TM5, D150A, zararlı mutasyon oligomerize dizisi yeteneğini azaltır. LMP-1 TM1 önemli ölçüde oligomerize değildir ve sadece boş olmayan dönüştürülmüş FHK12 hücreler için sinyal üzerinde çok düşük Miller Birim sinyali sergiliyor. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Şekil 1" /> Şekil 1. Karikatür ToxR muhabiri tahlil tasvir. Transmembran etki alanı (TMD) ToxR ve LacZ transkripsiyon aktivasyonu dimerization oligomerization sonuçları tahrik. LacZ gen ürünü, β-galaktosidaz oligomerize bir TMD eğilimi bir önlem olarak belirlenebilir. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Şekil 2" /> Şekil 2 ışık emici türleri o nitrophenolate (ONP) üretimi β-galaktosidaz sonuçları ONPG hidrolitik bölünme. <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Şekil 3" /> Şekil 3 pToxR7 Plazmid harita. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Şekil 4" /> Şekil 4 Temsilcisi ToxR transkripsiyonel muhabiri tahlil latent membran protein-1 transmembran etki alanı oligomerization eğilimi analiz. Transmembran etki alanı 1 (TM1) sadece zayıf bir etkileşim gösterir iken Transmembran etki alanı 5 (TM5), güçlü bir oligomerizes. Mutasyon D150A TM5 içinde oligomerize kabiliyetini önemli ölçüde azaltır. GPA, güçlü dimerization bir pozitif kontrol dizisi olarak yer almaktadır. Boş araştırıcıların bulgularıyla FHK12 hücreleri temsil eder. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Şekil 5" /> Şekil 5 protein ekspresyonu için Western Blot. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Şekil 6" /> Şekil 6 PD28 tamamlama tahlil periplasm doğru membran ekleme kontrol etmek için. Negatif kontrol, maltoz bağlayıcı protein eksikliği bir yapı gösterir.

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ToxR transkripsiyonel muhabiri tahlil oligomerize potansiyeline sahip transmembran dizileri tanımlamak için uyduruk bir yoludur. Bakteriyel iç zarı içinde meydana gelen etkileşimler olduğundan, bu testte membran-mimetik ortamlarda eğitim sistemlerinin geçerliliği ile ilgili konular circumvents. Paralel olarak tek bir plazmid birden çok TMDS klonlama kolayca yapılabilir ve tüm testi 96-plaka biçiminde yapılabilir göz önüne alındığında, bu testte yüksek verimlilik analizi için çok sayıda protein ...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> BamHI restriksiyon enzimi </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen enzimler alternatif tedarikçiler daha verimli olduğu tespit edildi </td></tr><tr><td> NheI restriksiyon enzimi </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen enzimler alternatif tedarikçiler daha verimli olduğu tespit edildi </td></tr><tr><td> 15 ml kültür tüpleri </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC medya </td><td> Teknova </td><td> S0225 </td><td> Uygun hacmi kadar yapılan ve otoklav ile sterilize. </td></tr><tr><td> LB medya </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Uygun hacmi kadar yapılan ve otoklav ile sterilize. </td></tr><tr><td> Kloramfenikol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Dondurucuda saklanan etanol içinde 30 mg / mL stok çözelti </td></tr><tr><td> Arabinoz </td><td> Fluka </td><td> 10839 </td><td> Dondurucuda saklanan su,% 2,5 (w / v) stok solüsyonu </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt Kimyasallar </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Sodyum dodecylsulfate (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-nitrofenil β-D-galactopyranoside (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-buffer </td><td></td><td></td><td> 16.1 g Na 2 HPO 4 5.5g NaH 2 PO 4 0.75g KCl 0.246g MgSO 4 1 lt, pH 7.0 Makyaj </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 ml β-mercaptoethanol, 2 mL kloroform, Z-buffer ile 20 ml kadar yapmak. 800 rpm&#39;de 1 dakika için 1 dakika, santrifüj Vortex. Her plaka için taze olun. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS 10 ml Z-tampon içinde çözünmüş </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg 10 ml Z-tampon ONPG. Her plaka için taze olun </td></tr><tr><td> ß-mercaptoethanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP monoklonal antikor (HRP konjuge) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Maltoz ile Minimal medya </td><td></td><td></td><td> 1 x M9 tuzlar,% 0.4 maltoz, 1 mg / ml tiamin, 2 mM MgSO 4 </td></tr><tr><td> 96-düz alt plakası </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Levha okuyucu </td><td> Beckman Coulter </td><td> DTX880 Multimode Dedektörü </td><td></td></tr><tr><td> Su banyosu </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Inkübatör Çalkalamalı </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

Çürütülmüştür beri protein transmembran etki basitleştirilmiş görünüm olarak sadece fosfolipid bilayers çapa uzun. Birçok durumda membran kapsayan proteinler eylem oldukça sofistike mekanizmaları gelişmiştir. Membran proteinleri, yapıları ve işlevleri modüle 1-3 hangi bir yolu yapılandırılmış transmembran oligomerler oluşturan, hidrofobik heliksler ve özel direkt temas 4,5 Kadar son tercihen sulu çözelti ve sürücü protein dernek. 6,7 Bununla birlikte, transmembran proteinlerinin etki moleküler tanıma çalışmaları hala gerisinde kalıyor bu farklı moleküller arası kuvvetler karşılaştırma membran ortamda bulunan amino asitlerin dağıtım odaklandı suda çözünen bölgeler. Büyük bir engel kalır: transmembran oligomerization elde edebilirsiniz olağanüstü spesifite ve afinite rağmen, kendi dernek 8 doğrudan ölçümü zordur. Integral membran proteini fonksiyon çalışmaya uygulanan geleneksel metodolojiler inceleme altında dizilerinin doğasında çıkılmazlık engel olabilir. Transmembran etki alanlarını temsil eden sentetik peptidlerin inceleyerek elde Biyofiziksel anlayışlar yararlı yapısal içgörü sağlayabilir. Ancak, deterjan misel veya hücre zarının taklit etmek için bu çalışmalarda kullanılan lipozom sistemleri biyolojik önemi sık sık sorgulanmaktadır; peptidler, bu koşullar altında anadili gibi bir yapı benimsiyor ve bunların fonksiyonel davranış, gerçekten yerli bir membran içinde eylem türü yansıtıyor mu ? Transmembran dizilerinin doğal fosfolipid bilayers etkileşimleri incelemek üzere, Langosch laboratuar ToxR transkripsiyonel muhabiri deneyleri geliştirdi. 9 transmembran ilgi alanı periplasm ve ToxR konumu, maltoz bağlayıcı protein ile bir kimerik protein olarak ifade edilir bir rapor sunmak oligomerization düzeyi (Şekil 1). Son on yıl içinde, diğer bazı gruplar (örneğin Engelman, DeGrado, Shai) daha da optimize ve bu ToxR muhabiri tahlil uygulanan 10-13 çeşitli ToxR deneyleri, hücre zarlarında protein-protein etkileşimleri test etmek için bir altın standart haline gelmiştir . Biz burada öncelikle Langosch tarafından geliştirilen protokoller aşağıdaki laboratuvarda yapılan tipik bir deneysel çalışma göstermektedir. Bu genellikle uygulanan yöntem E. transmembran alan öz-dernek analizi için yararlı coli, β-galaktosidaz üretim TMD oligomerization eğilimi değerlendirmek için kullanılır. TMD indüklenen dimerization üzerine, ToxR β-galaktosidaz LacZ gen up-regülasyonu neden ctx organizatörü bağlar. Kolorimetrik okuma kazandaki süreçlerin hücreleri ONPG eklenmesi ile elde edilir. Işık emici türleri o nitrophenolate (ONP) (Şekil 2) üretimini β-galaktosidaz sonuçları ONPG hidrolitik bölünme.

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Tek-geçişli transmembran etki oligomerization eğilimi (TMDS) değerlendirmek için etkili bir prosedür açıklanmıştır. E. coli muhabiri zorlanma ToxR için erimiş TMD oluşan Kimerik proteinleri ifade edilir. TMD-kaynaklı oligomerization ToxR, transkripsiyon ve muhabir protein üretimi,-galaktosidaz aktivasyonu dimerization neden olur.

ToxR Assay tarafından belirlenen Transmembran Domain Oligomerization Eğilimi

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

transmembrane-domain-oligomerization-propensity-determined-toxr

Research

JoVE Journal

Biology

15.1K Görüntüleme.  University of Colorado at Boulder. Tek-geçişli transmembran etki oligomerization eğilimi (TMDS) değerlendirmek için etkili bir prosedür açıklanmıştır. E. coli muhabiri zorlanma ToxR için erimiş TMD oluşan Kimerik proteinleri ifade edilir. TMD-kaynaklı oligomerization ToxR, transkripsiyon ve muhabir protein üretimi,-galaktosidaz aktivasyonu dimerization neden olur.

Video: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Yılında Kistik Fibrozis Transmembran İletkenlik Regülatörü Protein İfadesi ve Arıtma Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Biyoloji

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Olarak ifade edilmiştir kistik fibroz transmembran kondüktans regülatörü Protein Saflaştırılması<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Bir floresan bazlı Kalsiyum Mobilizasyon Deneyi G proteini ile birleştirilmiş alıcıların karakterizasyonu

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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).

Yaşayan Hücrelerde Protein-fragmanı tamamlama Assay (PCA) ile genom Protein-protein etkileşimi Eleme

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

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Bir transmembran proteinin yeniden oluşturulması, Mikroskop ve yama kelepçe Çalışmaları Dev tek-katmanlı keseler içine Voltaj kapılı iyon kanalı, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Intra-Sarkoplazmik Retikulum Ca Optik Haritalama<sup&gt; 2+</sup&gt; Ve Langendorff-perfüze Tavşan Heart Transmembran Potansiyeli

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Genetik ve biyokimyasal Yaklaşımlar<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Protein oligomerization Değerlendirilmesi: Riyanodin Reseptör Vaka Çalışması

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Endoplazmik retikulum ve mitokondri Etkileşimleri Çalışma ile<em&gt; In Situ</em&gt; Proximity ligasyon Testi Sabit Hücrelerde

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...