Deutsche Forschungsgemeinschaft (DFG) hibesi BE1911/8-1'e, LOEWE projesi GLUE'ya ve işbirlikçi araştırma merkezi Transport and Communication across Membranes'a (SFB807) finansal destek için teşekkür ederiz.

<ol>
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Yazarların açıklayacak hiçbir şeyleri yoktur.

CF ekspresyonunu optimize etmek için kurulum ve stratejiler ve fonksiyonel milletvekillerinin nanodisklere birlikte translasyonel olarak yerleştirilmesi açıklanmaktadır. Gerekli ekipman bir biyoreaktör, bir Fransız pres cihazı veya benzeri, bir UV / VIS ve floresan okuyucu, iki bölmeli bir konfigürasyon kurulumu için uygun CF reaksiyon kapları ve sıcaklık kontrollü bir inkübatörden oluşur. Diğer standart ekipmanlar, E. coli hücrelerinin toplanması için santrifüjlerin yanı sıra S30 lizatl...

Membran proteinleri (MPS), sulu ortamlarda çözünmemeleri nedeniyle protein üretim çalışmalarında zorlu hedeflerdir. Geleneksel MP üretim platformları, E. coli, maya veya ökaryotik hücreler gibi hücre tabanlı sistemlerden oluşur. Sentezlenen rekombinant milletvekilleri ya hücre zarlarından ekstrakte edilir ya da inklüzyon cisimciklerinden yeniden katlanır1. Deterjan çözünürlüğünden sonra milletvekilleri, in vitro sulandırma protokolleri oluşturularak uygun membran ortamlarına aktarılabilir. Veziküller ve lipozomların yanı sıra, nanodiskler2 veya salipro3 parçacıkları şeklinde düzlemsel membranlara MP resuentasyonu son zamanlarda rutin teknikler haline gelmiştir. Bununla birlikte, tüm bu stratejiler, milletvekilleriyle deterjan temasının istikrarsızlaşmaya, oligomerlerin ayrışmasına ve hatta protein yapısının ve aktivitesinin kaybına neden olabileceği anlamınagelir 4. Bu nedenle optimum deterjan çözünürlüğü ve sulandırma koşullarının taranması sıkıcı ve zaman alıcı olabilir5.
Hücresiz (CF) sistemlerin açık doğası, ekspresyon reaksiyonunun, tanımlanmış bir lipit bileşimine sahip önceden oluşturulmuş membranlarla doğrudan sağlanmasını sağlar. Bu lipit bazlı ekspresyon modunda (L-CF), sentezlenmiş milletvekilleri, sağlanan çift katmanlı 6,7'ye birlikte translasyonel olarak ekleme fırsatına sahiptir (Şekil 1). Düzlemsel lipid çift katmanlı disk8'i çevreleyen bir membran iskele proteininden (MSP) oluşan nanodiskler, bu strateji 9,10 için özellikle uygun görünmektedir. Nanodiskler rutin olarak çeşitli farklı lipitlerle in vitro olarak monte edilebilir, çok kararlıdırlar ve stoklar 1 mM'ye kadar konsantre edilebilir. Bununla birlikte, E. coli'de MSP ekspresyonu ve saflaştırılması gereklidir. Alternatif bir strateji olarak MSP, lipozomlarla beslenen KF reaksiyonlarında hedef MP ile birlikte eksprese edilebilir11,12,13. Hem MSP hem de MP için DNA şablonları reaksiyona eklenir ve ifade üzerine MP / nanodiskler oluşabilir. MSP üretiminden kaçınılırken, birlikte ekspresyon stratejisi, nihai DNA şablon konsantrasyonlarının dikkatli bir şekilde ince ayarlanmasını gerektirir ve numune üretiminin verimliliğinde daha yüksek varyasyonlar beklenebilir.
Milletvekillerinin önceden oluşturulmuş nanodisklerin zarlarına ko-translasyonel olarak eklenmesi, translokon makinelerinden ve sinyal dizileri13,14,15,16'dan bağımsız, fizyolojik olmayan ve hala büyük ölçüde bilinmeyen bir mekanizmadır. Yerleştirme etkinliğinin başlıca belirleyicileri, membran proteininin tipi ve sağlanan membranın lipit bileşimidir ve negatif yüklü lipitler için sıklıkla tercih edilir15,17. Nanodisk membranları nispeten sınırlı boyuttadır, MP yerleştirme18 üzerine önemli miktarda lipit salınır. Nanodisk boyutunun değişimi, daha yüksek oligomerik MP komplekslerinin 15,18'in yerleştirilmesini ve ayarlanmasını sağlar. Diğerlerinin yanı sıra, iyon kanalı KcsA, iyon pompası proteorhodopsin (PR) ve çoklu ilaç taşıyıcı EmrE15,18 için homooligomerik komplekslerin doğru montajı gösterilmiştir. Milletvekillerinin simetrik nanodisk membranına her iki taraftan da nispeten eşit frekansta girmeleri muhtemeldir. Bu nedenle, bir nanodiske yerleştirilen farklı monomerlerin veya oligomerlerin zıt yönelimlere sahip olabileceği düşünülmelidir. Bununla birlikte, oryantasyondaki bir önyargı, PR hexamers ve KcsA tetramerlerinin oluşumu için bildirilen işbirlikçi yerleştirme mekanizmalarından kaynaklanabilir18. MP oryantasyonundaki başka bir önyargı, muhtemelen nanodisk membranlarının kenarına yerleştirilmiş MP'lerin oryantasyon anahtarlarından kaynaklanabilir.
E. coli suşlarından CF lizat üretimi güvenilir bir rutin tekniktir ve hemen hemen her biyokimyasal laboratuvarda gerçekleştirilebilir19,20. Disülfit köprü oluşumunun yanı sıra, bir MP'nin E. coli lizatları kullanılarak sentezlenmesi durumunda, diğer birçok çeviri sonrası modifikasyonun bulunmadığı düşünülmelidir. Bu, yapısal çalışmalar için daha homojen örnekler üretebilirken, bireysel MP hedeflerinin işlevi üzerindeki potansiyel etkileri dışlamak gerekebilir. Bununla birlikte, E. coli CF lizatlarında G-protein kuplajlı reseptörlerin (GPCR) 14,21,22, ökaryotik taşıyıcıların 23 veya büyük heteromerik montajların 24'ünün yüksek kaliteli örneklerinin verimli bir şekilde üretilmesi, karmaşık hedefler için bile uygunluklarını göstermektedir. Bir numunenin hazırlık ölçeği miktarları (≈ 1 mg/mL), bir reaksiyon karışımı (RM) ve bir besleme karışımı (FM) bölmesinden oluşan iki bölmeli sürekli değişim hücresiz (CECF) konfigürasyonu ile elde edilebilir. FM hacmi, RM hacmini 15 ila 20 kat aşıyor ve düşük moleküler ağırlıklı enerji bileşikleri ve öncülleri19'dan oluşan bir rezervuar sağlıyor. Böylece reaksiyon ifadesi birkaç saat uzatılır ve MP hedefinin nihai verimi artırılır. RM ve FM bölmeleri, 10-14 kDa kesimli bir diyaliz membranı ile ayrılır. İki bölme, CECF reaksiyon kabının özel bir tasarımını gerektirir (Şekil 1). FM'i tutan özel pleksiglas kaplar ile birlikte RM kapları olarak ticari diyaliz kasetleri uygun örneklerdir. MP verimleri, RM: FM oranlarını değiştirerek veya belirli bir inkübasyon süresinden sonra FM'yi değiştirerek daha da manipüle edilebilir.
Bir MP'nin verimi ve kalitesi sıklıkla reaksiyon parametrelerinin yoğun optimizasyonunu gerektirir. CF ifadesinin önemli bir avantajı, bir MP'nin bireysel gereksinimlerine göre hemen hemen her bileşiği değiştirme ve ince ayar yapma olasılığıdır. Basit bir strateji, önce temel bir üretim protokolü oluşturarak bir MP'nin verimini artırmaya odaklanmak ve daha sonra reaksiyon ve katlama koşullarına ince ayar yaparak MP kalitesini optimize etmektir. KF lizatlarında fizyolojik süreçlerin olmaması ve karmaşıklığının azalması, milletvekillerinin verimli üretimi için yüksek başarı oranlarına neden olmaktadır25. DNA şablon tasarımı ve Mg2 + iyon konsantrasyonunun optimizasyonu için rutin hususlar çoğu durumda tatmin edici verimler elde etmek için yeterlidir26. İfade moduna bağlı olarak, MP kalitesinin optimizasyonu zaman alıcı olabilir, çünkü daha çeşitli parametrelerintaranması gerekir 14,17,22.
Tanımlanan CF ekspresyon platformunu oluşturmak için, E. coli CF lizat (i), T7 RNA polimeraz (ii), nanodiskler (iii) ve temel CECF reaksiyon bileşiklerinin (iv) üretimi için protokoller gereklidir (Şekil 1). E. coli K12 suşu A1927 veya BL21 türevleri, verimli S30 (30.000 x g'de santrifüjleme) lizatlarının hazırlanmasında sıklıkla kullanılır. S30 lizatlarının yanı sıra, diğer g-kuvvetlerinde (örneğin S12) santrifüj edilmiş karşılık gelen lizatlar da kullanılabilir. Lisatlar ekspresyon verimliliği ve proteom karmaşıklığı bakımından farklılık gösterir. Tanımlanan protokol ile hazırlanan S30 lizatının proteomu28 ayrıntılı olarak incelenmiştir. S30 proteomu hala iyon kanallarının ekspresyonu ve analizi üzerine arka plan problemleri verebilecek bazı artık dış zar proteinleri içerir. Bu tür hedefler için S80-S100 lizat kullanılması önerilir. Lisat hazırlama sırasında değerli bir modifikasyon, hücrelerin orta log büyüme fazında kombine ısı şoku ve etanol beslemesi ile SOS yanıtının indüklenmesidir. Elde edilen HS30 lizatları şaperonlar bakımından zenginleştirilmiştir ve gelişmiş MP katlama22 için S30 lizatlarla karışımlarda kullanılabilir. E. coli lizatlarındaki CF ekspresyonu, T7 RNA polimeraz (T7RNAP) tarafından kontrol edilen promotörler içeren DNA şablonları ile birleştirilmiş bir transkripsiyon / translasyon işlemi olarak çalıştırılır. Enzim, pAR1219 plazmid29'u taşıyan BL21 (DE3) Yıldız hücrelerinde verimli bir şekilde üretilebilir.
MSP1E3D1'in iki kopyası, 10-12 nm çapında bir nanodiskte toplanır, ancak aşağıda açıklanan protokol diğer MSP türevleri için de çalışabilir. Bununla birlikte, MSP1E3D1 ile oluşturulanlardan daha büyük nanodiskler daha az kararlı olma eğilimindeyken, MSP1 gibi MSP türevleriyle oluşturulan daha küçük nanodiskler daha büyük MP'leri veya MP komplekslerini barındırmayabilir. MSP1E3D1 nanodiskleri, çok çeşitli farklı lipitler veya lipit karışımları ile in vitro olarak monte edilebilir. Önceden oluşturulmuş nanodiskler genellikle -80 ° C'de 1 yıl > stabildir, stabilite ise farklı lipit bileşenleri için değişebilir. Bir MP'nin katlanması ve stabilitesi üzerindeki lipit etkilerinin taranması için, temsili çeşitli lipitler / lipit karışımları ile monte edilmiş kapsamlı bir nanodisk seti hazırlamak gerekir. Aşağıdaki lipitler iyi bir başlangıç seçimi sağlayabilir: 1,2-Dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) ve 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC).
3 mL CECF reaksiyonunun hazırlanması için bir protokol tanımlanmıştır. 1: 1 oranında daha fazla yukarı veya aşağı ölçeklendirme mümkündür. İki bölmeli CECF konfigürasyonu için, tüm bileşikleri içeren bir RM ve sadece düşük moleküler ağırlıklı bileşikleri içeren bir FM hazırlanmalıdır. 10-14 kDa MWCO'lu ticari 3 mL diyaliz cihazları RM konteyner olarak kullanılabilir ve daha sonra FM'i tutan özel yapım pleksiglas kaplara yerleştirilir (Şekil 1D)30. RM: FM oranı 1: 20'dir, bu nedenle 3 mL RM için 60 mL FM hazırlanmalıdır. DNA şablonları yayınlanan kılavuzlara göre hazırlanmalıdır ve hedefin okuma çerçevesinin kodon optimizasyonu ürün verimini daha da artırabilir26. Hazırlık ölçeği CECF reaksiyonu için, PR üretimi için belirlenmiş bir protokol tanımlanmıştır. Yeni milletvekilleri için ifade protokolleri oluşturmak için, verimi ve kaliteyi artırmak için genellikle belirli bileşiklerin optimizasyon ekranlarını (Tablo 1) gerçekleştirmek gerekir. Mg2 + iyonları için, farklı DNA şablonları için sıklıkla önemli varyasyonlar gösteren iyi odaklanmış bir konsantrasyon optimumluğu vardır. Yeni T7RNAP veya S30 lizat partileri gibi diğer CF reaksiyon bileşikleri, optimal Mg2 + konsantrasyonunu daha da değiştirebilir. Örnek olarak, 14-24 mM konsantrasyon aralığında ve 2 mM'lik adımlar halinde tipik bir Mg2+ ekran tanımlanmıştır. Her konsantrasyon kopyalar halinde ve analitik ölçekte CECF reaksiyonlarında taranır. CECF reaksiyon kabı olarak, RM'yi tutan özel yapım Mini-CECF Pleksiglas kaplar30, FM'yi tutan standart 24 delikli mikro plakalarla birlikte kullanılır (Şekil 1B). Alternatif olarak, uygun kurulumlarda 96 derin kuyucuklu mikroplakalar veya diğer ticari diyalizör cihazları ile birlikte ticari diyaliz kartuşları kullanılabilir (Şekil 1C).

<table>
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (DMPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840445P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850345C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840475C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (POPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)</td>
			<td>Carl Roth (Germany)</td>
			<td>4855</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>4227</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth (Germany)</td>
			<td>9781</td>
			<td></td>
		</tr>
		<tr>
			<td>[3H]-dihydroalprenolol Hydrochloride</td>
			<td>American Radiolabeled Chemicals (USA)</td>
			<td>ART0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl phosphate lithium potassium salt (ACP)</td>
			<td>Merck (Germany)</td>
			<td>1409</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate (ATP)</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr>
			<td>Alprenolol hydrochloride</td>
			<td>Merck (Germany)</td>
			<td>A0360000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange chromatography column material: Q-sepharose&#174;</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>Q1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Type GE 446EC-1</td>
			<td>Gettinge (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor Type 884 124/1</td>
			<td>B.Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid</td>
			<td>Carl Roth (Germany)</td>
			<td>8137</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R250</td>
			<td>Carl Roth (Germany)</td>
			<td>3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks 500 ml baffled flasks, 2 l baffled flasks</td>
			<td>Schott Duran (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate disodium salt</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>C1506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose monohydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6780</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassiumhydrogen phosphate trihydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing SpectrumTM Labs Spectra/PorTM 12-14 kD MWCO Standard RC tubing</td>
			<td>Fisher Scientific (Germany)</td>
			<td>8700152</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreit</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell disruptor</td>
			<td>SLM; Amico Instruments (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth (Germany)</td>
			<td>3783</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate di-sodium salt (GTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>K025</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAC column: HiTrap IMAC HP 5 mL</td>
			<td>GE Life Sciences (Germany)</td>
			<td>GE17-5248</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth (Germany)</td>
			<td>3899</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td>Carl Roth (Germany)</td>
			<td>2316</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Carl Roth (Germany)</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Carl Roth (Germany)</td>
			<td>3076.1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Carl Roth (Germany)</td>
			<td>HN23</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>T202</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Carl Roth (Germany)</td>
			<td>T203</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>3774</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Carl Roth (Germany)</td>
			<td>3772</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine</td>
			<td>Carl Roth (Germany)</td>
			<td>3187</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Carl Roth (Germany)</td>
			<td>3852</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Carl Roth (Germany)</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Carl Roth (Germany)</td>
			<td>1699</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Carl Roth (Germany)</td>
			<td>4207</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Carl Roth (Germany)</td>
			<td>9359</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Carl Roth (Germany)</td>
			<td>1713</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Carl Roth (Germany)</td>
			<td>1709</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Carl Roth (Germany)</td>
			<td>4682</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Carl Roth (Germany)</td>
			<td>1738</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophane</td>
			<td>Carl Roth (Germany)</td>
			<td>7700</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Carl Roth (Germany)</td>
			<td>T207</td>
			<td></td>
		</tr>
		<tr>
			<td>MD100 dialysis units</td>
			<td>Scienova (Germany)</td>
			<td>40077</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2-Hydroxyethylpiperazine-N&#39;-2-ethansulfonic acid (HEPES)</td>
			<td>Carl Roth (Germany)</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecylphosphocholine</td>
			<td>Antrace (USA)</td>
			<td>F308S</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE chamber: Mini-Protean Tetra Cell</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel casting system: Mini Protean Handcast systems</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel power supply: Power Pac 3000</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone/peptone from caseine</td>
			<td>Carl Roth (Germany)</td>
			<td>6681</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump: ip-12</td>
			<td>Ismatec (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenol pyruvate monopotassium salt</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>860077</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth (Germany)</td>
			<td>P018</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Carl Roth (Germany)</td>
			<td>4986</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate Kinase</td>
			<td>Roche (Germany)</td>
			<td>10109045001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter: Hidex 300 SL</td>
			<td>Hidex (Finland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth (Germany)</td>
			<td>8029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>K305</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>P029</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Nanodrop</td>
			<td>Peqlab (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer/fluorescence reader Spark&#174;</td>
			<td>Tecan (Switzerland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (E. coli)</td>
			<td>Roche (Germany)</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra sonificator</td>
			<td>Labsonic U, B. Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#8217;-triphosphate tri-sodium salt (UTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>U6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-30 antifoam</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>A6457</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Carl Roth (Germany)</td>
			<td>2904</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-translational insertion of membrane proteins into preformed nanodiscs

Hücresiz ekspresyon sistemleri, membran proteinleri gibi karmaşık proteinlerin bile işlevsel katlanmasını desteklemek için reaksiyon ortamlarının özel tasarımına izin verir. Membran proteinlerinin nanodiskler olarak tedarik edilen önceden oluşturulmuş ve tanımlanmış membranlara ko-translasyonel olarak yerleştirilmesi ve katlanması için deneysel prosedürler gösterilmiştir. Protokol tamamen deterjansızdır ve bir gün içinde miligram saflaştırılmış numune üretebilir. Elde edilen membran proteini / nanodisk örnekleri, çeşitli fonksiyonel çalışmalar ve kristalizasyon, nükleer manyetik rezonans veya elektron mikroskobu gibi yapısal uygulamalar için kullanılabilir. Hücresiz lizatlar, tasarlanmış membranlı nanodiskler, kritik stok çözeltileri ve iki bölmeli hücresiz ekspresyon reaksiyonlarının montajı gibi temel anahtar bileşenlerin hazırlanması açıklanmaktadır. Membran proteinlerinin katlanma gereksinimleri çok çeşitli olabileceğinden, bu protokolün ana odak noktası, kritik temel reaksiyon bileşikleri, nanodisklerin membran bileşimi, redoks ve şaperon ortamı veya DNA şablon tasarımı gibi numune kalitesi için önemli olan parametrelerin ve reaksiyon adımlarının modülasyonudur. Tüm süreç proteorhodopsin ve bir G-protein eşleşmiş reseptör sentezi ile gösterilmiştir.

İnce ayar reaksiyon bileşiklerinin, sentezlenmiş milletvekillerinin nihai verimi veya kalitesi üzerindeki etkisi örneklendirilmiştir. Sık kullanılan rutin bir yaklaşım, CF reaksiyonlarında optimal Mg2+ konsantrasyonunu, sistem verimliliğinin uygun bir monitörü olarak yeşil floresan proteini (GFP) ekspresyonu ile ayarlamaktır. Örnek olarak, GFP, 14 ila 24 mM arasındaki Mg 2+ konsantrasyonlarında bir pET-21a (+) vektöründen sentezlendi (Şekil 2). SDS...

Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Önceden oluşturulmuş nanodisklere birlikte translasyonel yerleştirme, tanımlanmış lipit ortamlarında deterjanlarla temas etmeden hücresiz sentezlenmiş membran proteinlerinin incelenmesini mümkün kılar. Bu protokol, temel sistem bileşenlerinin hazırlanmasını ve ifade verimliliğini ve numune kalitesini artırmak için kritik parametreleri açıklar.

Membran Proteinlerinin Önceden Oluşturulmuş Nanodisklere Ko-Translasyonel Olarak Yerleştirilmesi

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

co-translational-insertion-membrane-proteins-into-preformed

Research

JoVE Journal

Biochemistry

3.2K Görüntüleme.  Goethe University Frankfurt. Önceden oluşturulmuş nanodisklere birlikte translasyonel yerleştirme, tanımlanmış lipit ortamlarında deterjanlarla temas etmeden hücresiz sentezlenmiş membran proteinlerinin incelenmesini mümkün kılar. Bu protokol, temel sistem bileşenlerinin hazırlanmasını ve ifade verimliliğini ve numune kalitesini artırmak için kritik parametreleri açıklar.

Video: Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

İskele Lipozomlar kullanarak Lipid-proksimal protein-protein etkileşimleri Sulandırın için<em&gt; İn Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Biyokimya

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Yöntem gözünüzde canlandırın ve Transmisyon Elektron Mikroskobu Membran Etkileşen Proteinler Analiz için

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

Azot kavitasyon ve diferansiyel Santrifüjü sağlar periferik membran proteinlerinin kültürlü hücrelerdeki dağılımını izlemek için

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Deterjan-Alerjik membran proteinlerinin Ultrafast sulandırma tamamlayıcı-Proteoliposomes ücret Fusogenic kullanarak Lipid Bilayers içine.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Membran proteinlerinin Hofstede deterjan duyarlı tespiti

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Bir floresan dalgalanma spektroskopisi tahlil Protein-Protein etkileşimlerinin hücre-hücre kişiler

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick, Aerodinamik Protein Bağlayıcı Tahliller için Biyotinylated Peptidiskler içine Membran Proteinlerin Bir Adım Dahil

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Beyin Zarı Proteinlerinin Palmitoylarasyonunun Kantitatif Tayini için Acyl-PEGyl Exchange Jel Shift Tayini

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Sağlam, fonksiyonel membran proteinlerine bağlanan nükleotidin gerçek zamanlı olarak ölçülmesi

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Olgun Pirincin Enine Kesiti (Oryza sativa L.) Hareketsizleştirme Desteği Olarak Pipet İpuçlarını Kullanarak Elektron Mikroskopi Görüntülemesini Taramak için Çekirdekler