Name: synthesis of protein bioconjugates via cysteine-maleimide chemistry
Uploaded: 2016-07-20T15:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.

Not: Şekil 1 &#39;de gösterildiği gibi, aşağıdaki protokolü, bir protein-boya Bioconjugate sentezi için tasarlanmıştır notları yerleştirilmiş membran protein ile yardımcı olmak için durumlarda bu, serbest yüzey sistein ihtiva eden proteinlerle bir maleimid reaksiyonu için genel bir protokoldür. Bioconjugatlar protein polimer Bioconjugatlar ve sentetik protein dimeri (protein-protein) Bioconjugatlar. Bu özel durumda, protein izo-1 sitokrom C yüksek ölçüde özel bir işaretleme meydana sağlar tepkimeye uygun bir yüzey sistein artığı bulunur. ilgi konusu bir protein çoklu sisteyin artıkları ihtiva ediyor ise, aynı protokol özgüllük ve ürün homojenliği kaybı olsa geçerlidir. Özgüllük gerekli değilse N -hydroxysuccinimidyl esterleri ya da izotiosiyanatlar kullanılarak Kimya hedefleme yüzeyi lisin artıkları, daha basit bir yaklaşım olabilir. <img alt="Şekil 1" src="/files/ftp_upload/54157/54157fig1.jpg" /> <strong&gt; Şekil 1 Bioconjugation Reaksiyon Şeması. Örnek durumda, hafif hasat olarak rutenyum esaslı bir anteni molekülü (CYS102) rutenyum esaslı bir anteni molekül üzerinde bir asılı maleimid ve açık bir sistein kalıntısının Michael ilave edilerek sitokrom c bağlı olacaktır protein. Cyt c yüzeyinin kırmızı alan heme grubu gösterir. <a href="https://www.jove.com/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Sitokrom c 1. Saflaştırma Not: Bu adım, tüm proteinlerin için geçerli değildir. Bununla birlikte, ticari bir kaynaktan elde edilen bir protein, bir daha arıtılmadan 13 çıkarılabilir gereken diğer istenmeyen protein izoformları içeren olduğunu bilmek önemlidir. <ol><li> Pr ultra saf su, 1 L (= 120 g mol-1 W E) sodyum dihidrojen fosfat 2.4 g çözülür20 mM NaH 2 PO 4 ihtiva eden bir tampon çözelti epare. pH 7-1 M NaOH ile pH ayarlayın. Not: Bu protokolde kullanılan fosfat tampon her gün taze olarak hazırlanmıştır, ve kullanımdan önce 0.2 um&#39;lik bir selüloz zar filtresi gerekir. </li><li> 20 mM NaH 2 PO 4 ve 1 M NaCl tampon yapmak için sodyum klorid 29.22 gramını (ağırlık M = 58.44 g mol-1), 20 mM NaH 2 PO 4 tampon, 500 ml içinde çözülür. Bu adım 1.6) &#39;de arıtma için elüsyon tamponudur. </li><li> PH 7 20 mM fosfat tamponu içinde 6 ml liyofilize sitokrom c (cyt c) 12.0 mg eritin. </li><li> Ayrıca, bir 1 M stok çözelti hazırlamak için ultra saf su 95,3 ul ditiyotreitol 14.7 mg (DTT, B, E = 154,25 g / Mol) çözülür. Not: bu madde, sulu çözelti içinde oksidatif deaktivasyon duyarlı olduğu gibi DTT stok çözeltisi taze hazırlanmalıdır. </li><li> ölmekProtein çözeltisi içine 1 M DTT çözeltisi 60 ul ette Cyt c azaltır. çözeltinin renk karıştırma üzerine ışık kırmızı koyu kırmızı değişecektir. </li><li> Herhangi bir kromatografik ortama enjekte etmeden önce 0.22 mikron düşük protein bağlayıcı PVDF şırınga filtresi aracılığıyla azaltılmış protein çözüm Filtre. </li><li> enjeksiyon döngüsü ve Hızlı protein sıvı kromatografi (FPLC) aletin UV-Vis detektörü ile 3.3 ml güçlü katyon değişim sütunu takın. </li><li> 1 ml / dk&#39;lık bir akış oranı kullanılarak, 20 mM pH 7 fosfat tamponunun 3 sütun hacim ardından ultra saf su, 3 kolon hacminde ile sütun dengelenmesi. </li><li> Yük 1 enjeksiyon döngü içine indirgenmiş ham cyt c ml ve 328 bir degrade yöntemi başlatmak - 5 sütun hacmi üzerinde 450 mM NaCl,. UV-Vis dedektör 280 nm ve 410 nm kanalı izlemek, ve en büyük zirve toplamak. </li><li> iso-2 sitokrom Zehir 1 M 2 kolon hacmi tuz konsantrasyonunu arttırmakE, C ve sütun boşaltılan sonra, aşağıdaki ham kısım enjekte edilmeden önce fosfat tampon 20 mM 2 sütun hacmi ile sütun yeniden dengeye. </li><li> tüm ham protein saflaştırılmış kadar tekrarlayın 1.9-1.10 adımları. </li><li> izo-1 ihtiva eden fraksiyonlar bir havuz ve 3.5 kDa moleküler ağırlık cutoff değeri (MWCO) sıkma filtre kullanılarak ve 3,000 x g de santrifüj bunları konsantre edin. </li><li> 3.5 kDa MWCO diyaliz kasetleri içine konsantre protein yükleyin ve su 2 değişikliklerle, ultra saf su bir gecede karşı dialyze. </li><li> 10 ul alan, aşırı saf su ile 100 ul&#39;ye seyreltilmesi, ve absorbans spektrumunun alınarak saf, konsantre protein çözeltisi konsantrasyonunu belirlemek. Düşük hacim kullanın, 100 ul kuvars küvet protein emme spektrumları elde etmek. 100 uM - Tipik olarak, seyreltilmemiş protein konsantrasyonu 50 mertebesindedir. <ol><li> Concentratio ölçmek için Cyt c karakteristik 410 nm tepe kullanınn 97.6 cm -1 mM -1 molar absorptivite değeri Beer-Lambert yasası kullanılarak: A = ε × c × ι Bir emme olduğu, ε molar emicilik, Cı mM konsantrasyonu, ve ι cm 13 küvetin yolu uzunluğudur. </li></ol></li><li> Bu noktada, 1 mL alikotlara protein bölmek ve gerekli olana kadar -20 ° C&#39;de dondurulmuş tutun. Sit c dondurulmuş ve yapı ya da fonksiyon kaybı olmadan çözülmüş, ancak tekrar döngüleri proteinin denatüre başlayacak edilebilir. Not: Proteinler farklı derecelerde dondurma tahammül. Örneğin, 5,9 dondurulamaz yeşil floresan protein yerine bir buzdolabı içinde depolanmıştır. </li></ol> Biyokonjugatlar c Sitokrom 2. Sentezi <ol><li> rutenyum 0.9 mg çözülür (II) bisterpyridine maleimide (Ru (II) (TPY) 2 -maleimid, 0.975 umol, 6 eşdeğer) ihtiva eden bir 600 ul. Not: kullanılan maleimid suda çözünür ise, su yerine asetonitril içinde bir stok çözeltisi hazırlanır. maleimid reaksiyon tamponu çözünür olması çok önemlidir. Dimetilsülfoksid, N, N-dimetilformamid ve asetonitril tüm yaygın düşük moleküler ağırlık 12, genellikle hidrofobik maleimid reaktiflerin çözünmesine yardımcı olmak için yardımcı maddeler kullanılmaktadır. </li><li> 20 mM&#39;ye seyreltilmiş EDTA çözünene kadar, tipik olarak (birkaç gram katı sodyum hidroksit ilave pH 7 olan 100 mM fosfat tamponu ve 100 mM etilendiamintetraasetik asit (EDTA, Mw = 292,24 g / mol) ihtiva eden bir tampon çözeltisi hazırlayın ) ile pH 7&#39;ye ayarlanır. </li><li> (286,65 g / Mol w = TCEP 15, m), aşırı saf su, 1 ml bir 10 mM stok çözelti hazırlamak için tris (2-karboksietil) fosfin hidroklorür 2.87 mg çözülür. Bu adım ithalatAnt protein sistein tam 15 bağlanması maleimide önceden indirgenmiş olduğundan emin olmak için. Not: bu madde, sulu çözelti içinde oksidatif deaktivasyon duyarlı olduğu TCEP stok çözeltisi, her bir reaksiyon için, taze hazırlanmalıdır. </li><li> 50 ml&#39;lik plastik bir tüp içinde 100 mM fosfat / EDTA stok çözeltisi 3 ml ultra saf su 11,4 ml birleştirin. Protein ve maleimid çözeltisi ile, daha da seyreltildi zaman tampon konsantrasyonunun 20 mM olacak. Not: etiketlendikten protein, bir trans membran proteini olup, uygun bir deterjan proteini çözünür hale getirmek için tampona eklenir sağlamak. bir deterjan kullanılmazsa zar proteini yavaş olumsuz reaksiyon verimlerini etki edecek ve, hızlandırabilir. Sonraki tüm saflaştırma aşamaları deterjan kullanın. </li><li> Fosfat-EDTA tampon maddesi ile arıtılmış, Cyt c 0.15 umol (50 uM çözelti 3 ml, 1 eşdeğer) eklenir. </li><li> TCEP stok SOLUT 7.5 ul ekleyinprotein çözeltisi iyon (0.075 umol, 0.5 eşdeğer) ve sistein oksidasyon nedeniyle dimerize olabilir herhangi bir protein azaltmak için 5 dakika boyunca karıştırıldıktan bırakın. Not: İlgili protein kolayca dimerize yoksa TCEP katmayın. indirgeyici olmayan koşullar altında, bir protein jeli çalıştırarak kontrol edin. </li><li> 24 saat süre ile, oda sıcaklığında, karanlıkta, Ru (II) (TPY) Cyt c düşük tamponlu solüsyona 2 -maleimid asetonitril çözeltisi 600 ul ekleyin ve reaksiyon karışımı, karıştırma bırakın. </li><li> 3.5 kDa MWCO döndürme filtreleri kullanarak reaksiyon karışımının, 3000 xg&#39;de santrifüj süzüntü berrak bitene kadar taze 20 mM fosfat tamponu ile 2-3 kez tekrar konsantre. arıtılmadan bir sonraki aşamada önce mümkün olduğu reaksiyon karışımından kadar tepkimemiş maleimid çıkarın. Not: Bu noktada ham bioconjugation Karışım, karanlıkta, buzdolabında saklanabilir. Centr ile reaksiyona girmeyen maleimid boya kaldırmak için önemlidirdepolamadan önce diyaliz ifuge maleimid yavaş olması ve spesifik olmayan protein yüzeyi üzerinde lizin kalıntıları ile reaksiyona girebilir. </li></ol> Sitokrom c Bioconjugatlar 3. saflaştırılması <ol><li> 20 mM NaH 2 PO 4 ve 0.5 M NaCl içeren bir tampon çözeltisi hazırlamak için, sodyum dihidrojen fosfat 2.4 g ve ultra saf su, 1 L sodyum klorür 29.22 g çözündürülür. Bu hareketsizleştirilmiş metal afinite kromatografisi (IMAC) saflaştırma için çalışan tampondur. </li><li> Imidazol 17 g (ağırlık M = 68,077 g mol-1), 20 mM NaH 2 PO 4 ve 0.5 M NaCl tampon 500 ml içinde çözülür. Bu IMAC arıtma için elüsyon tamponu olduğunu. </li><li> 1 M NaOH veya HCI kullanılarak pH 7&#39;ye Her iki tampon maddesi pH ayarlama, ve FPLC kullanılmadan önce 0.22 um rejenere selüloz membranlar yoluyla filtre. </li><li> Satın alınan IMAC sütun col yüklenen herhangi bir metal iyonları olmadan sevk edilir olarakUMN, ultra saf su 3 ultra saf su ml, 100 mM nikel asetat çözeltisi, 3 ml ve 6 ml ile yıkanması ile Ni2 + merkezli saflaştırılması için kolon hazırlanmıştır. Kolon hemen kullanılmak üzere değilse% 20 3 ml etanol ile yıkama ve bakteri büyümesini engellemek üzere buzdolabı içinde sütun saklayın. </li><li> Bir Ni 2+ enjeksiyon döngü ve UV-Vis dedektör arasındaki FPLC 1 ml IMAC sütunu -yüklü takın. </li><li> 20 mM fosfat, 3 kolon hacminde, 0.5 ml / dakika bir akış oranı ile 0.5 M NaCl tampon madde ile sütundan dengelenmesi. </li><li> Yük Ni2 + IMAC kolonu (0.22 um&#39;lik bir şırınga filtreden filtre) ham tepkime karışımından 100 ul. Rutenyum bisterpyridine-sitokrom C biokonjugatlannı elüte etmek için 125 mM 3 üzerinde sütun hacim 0 ilâ bir imidazol gradyanı, ardından reaksiyona girmemiş Cyt c elüt edilmesi için çalışan tampon, 3 kolon hacminde ile kolon yıkaması (Ru (II) &#39;-cyt c) . </li><li> C yıkamaTüm ham Bioconjugate saflaştırılmıştır kadar olumn 5 kolon hacmi için 250 mM imidazol tampon maddesi ile, daha sonra 20 mM fosfat, 0.5 M NaCl ve adımı tekrar 3.7 ile kolon yeniden dengeye. </li><li> Bioconjugate kesirler havuzu ve 3.000 x g santrifüj, 3.5 kDa MWCO döndürme filtresi kullanarak konsantre. </li><li> 3.5 kDa MWCO diyaliz kasetleri içine konsantre biokonjugatlannı yükleyin ve su 2 değişikliklerle, ultra saf su bir gecede karşı dialyze. </li><li> Izo-1 sitokrom c ile aynı molar absorptivitesi değerini kullanarak, UV-Vis saf, konsantre edildi Bioconjugate çözeltisinin konsantrasyonu belirlemek (97.6 mm-1 cm-1) 410 nm&#39;de. 100 mcM - Tipik olarak, Bioconjugate konsantrasyonu 50 mertebesinde olduğunu. </li><li> 25 ul kısma biokonjugatlannı bölün ve ihtiyaç duyulan kadar -20 ° C&#39;de dondurulmuş tutun. </li></ol> Sitokrom c Bioconjugatlar 4. Karakterizasyonu <ol><li> caydırmak-TOF MS tarafından Bioconjugate kütlesinin ermesi <ol><li> bir asetonitril / su / trifloroasetik asit çözeltisi, 1 ml kafeik asit, 10 mg çözülür (80: 20: 0.1, v / v / v). </li><li> kafeik asit çözeltisi 5 ul derişik protein çözeltisi 5 ul seyreltilir. </li><li> Nokta 0.5 MALDI hedef plaka üzerine kafeik asit çözeltisi ul ve çözüm kurumasını bekleyin. </li><li> Nokta 0.5 Bu kafeik asit yerinde üstündeki numune / matris çözümü ul, spot kurumasını bekleyin. &quot;Sandviç&quot; matris katmanları arasındaki numunenin bu üstündeki numune matris başka 0.5 ul nokta ve kurumasını bekleyin. </li><li> Proteinlerin 16 için uygun olan alet ayarları kullanılarak lineer şekilde kütle spektrumları elde edin. </li></ol></li><li> Jel elektroforezi ile biyokonjugatlar incelenmesi <ol><li> Her protein örneğinin 10 ul önceden karıştırılmış lityum dodesil sülfat (LDS, pH 8.4) tamponu (4x) t ile protein örneklerinin seyreltilmesi tarafından çalıştırılacak hazırlayıned oyuk başına yaklaşık 20 ug nihai konsantrasyon. indirgeyici koşullar gerektiren kuyuları, 500 mM ditiyotreitol (DTT) madde (10x) azaltmak için 1 ul ekle. </li><li> 10 dakika boyunca 70 ° C&#39;de ısı örnekleri. </li><li> Jel Yürütme tamponu hazırlamak için önceden karıştırılmış, 50 ml, 1 M MES, 1 M Tris baz,% 2 SDS, 20 mM EDTA (pH 7.7), ultra saf su 950 ml ticari bir kaynaktan tampon karışımı (20x) çalıştıran ekleyin. </li><li> jelden plastik tarak çıkarın ve jel çalışan tankında jel yerleştirin. Kuyular tamponu ile kaplıdır, böylece jel çalışan tampon tankı doldurun. </li><li> Numuneleri dikkatli bir şekilde prefabrik% 12 Bis-Tris üzerine, 1 mm boyunda, pipet uçları kullanılarak 10 oyuklu jel yükleme yardımcı olmak için. analiz yardım etmek için jel orta kuyuya prestained 10 protein moleküler ağırlık markerine (3-188 kDa) yükleyin. </li><li> 35 dakika için 200 V sabit gerilimde jel üzerinde çalıştırıldı. </li><li> 2 saat süre ile, ticari bir Coomassie mavisi solüsyonu ile jel leke ve ultrap yıkayın48 saat boyunca ure su. </li></ol></li><li> UV-Vis spektroskopisi yoluyla Bioconjugate saflık tayini <ol><li> Ru (II) (TPY) 2 -maleimid, izo-1 Cyt C ve Ru (II) &#39;-cyt Cı 5 uM çözeltilerinin 120 ul hazırlayın. </li><li> 250 nm 650 nm, tek ultra saf su içeren kuvars küvette bazal spektrumunu ölçün. </li><li> Küvet durulandı ve her bir ölçüm arasında kurutulur sağlanması her bir bileşenin spektrumunu ölçün. </li><li> Dalga boyunun bir fonksiyonu olarak her bir bileşenin absorbans Konu ve 1 belirlemek için nihai ürün ile başlangıç ​​malzemelerinin lineer bir miktar karşılaştırın Ru (II), (TPY) Cyt c 2 -maleimid 1 oranında reaksiyona. </li></ol></li></ol>

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Chem. 11 (28), 4602-4612 (2013).</li><li>Signor, L., Boeri Erba, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+Laser+Desorption/Ionization+Time+of+Flight+(MALDI-TOF)+Mass+Spectrometric+Analysis+of+Intact+Proteins+Larger+than+100+kDa.">Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa.</a> J. Vis. Exp. , e50635 (2013).</li><li>Foucher, M., Verdi&#232;re, J., Lederer, F., Slonimski, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+a+non-trimethylated+iso-1+cytochrome+c+in+a+wild-type+strain+of+Saccharomyces+cerevisiae).">On the presence of a non-trimethylated iso-1 cytochrome c in a wild-type strain of Saccharomyces cerevisiae).</a> Eur. J. Biochem. 31, 139-143 (1972).</li><li>M&#252;ller, M., Azzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+beef+heart+cytochrome+oxidase+subunit+III+with+eosin-5-maleimide.">Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.</a> FEBS Lett. 184 (1), 110-114 (1985).</li><li>Shen, B. Q., 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=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat. Biotechnol. 30 (2), 184-189 (2012).</li></ol>

The authors have nothing to disclose.

Bir bioconjugation önce başlangıç ​​malzemelerinin saflaştırılması büyük önem taşımaktadır. Ticari rekombinan kaynaklardan elde edilen proteinler, sıklıkla farklı yüzey kimya ve reaktiviteye sahip, ilgilenilen protein, diğer izoformları içerir. Örneğin, tarif edilen bioconjugation olarak, ticari olarak temin edilebilir Cyt Cı iki izo-1 ve izo-2 Cyt C 12,14,17 oluşan bir karışımı içerir. İzo-2 ve izo-1 sitokrom c formları temel fark, izo-1 sitokrom...

Bioconjugation kovalent bir boya, bir ilaç veya bir polimer gibi bir sentetik molekülü bir ya da sadece bir biyomolekülün bağlantı içerir. Protein bioconjugation yöntemleri artık yoğun protein yapma, floresan boya etiketleme 1,2 arasında değişen uygulamalarla birçok kimya, biyoloji ve nanoteknoloji araştırma gruplarında kullanılan (antikor) 3 (antikor ilaç konjugatları - ADK) -prodrugs protein dimerleri 4,5 sentezini , nanotıp 8 ve sistemler kimya 9 kullanılan kendinden montaj protein polimer melezler 6,7 kadar. kimya Özelliği, her zaman kritik olmasa da, hedef proteinin aktif site ile müdahale etmeyecek şekilde, en fonksiyonel proteini biyokonjugatlar için büyük önem taşımaktadır, bioconjugation kullanılır. Ilgilenilen protein i) hedefleme nadir veya benzersiz siteler: İdeal bioconjugation reaksiyonu da dahil olmak üzere, çeşitli kriterleri yerine getirmek gerekiyorii) iii) açılımı protein önlemek için denatüre edici olmayan koşullar altında devam, bu hedefe yönelik seçici olmak ve hedef protein genellikle alt milimolar konsantrasyonda kullanılabilir olarak iv) yüksek verimli olacak. Maleimid - sistein Michael katılması, tüm bu kriterlere uygun yakın geliyor ve bu nedenle uzun Bioconjugate Chemistry 10 alanında özel bir statüye iddia için vardır. i) kendi yüzeyi üzerinde tek bir sistein kalıntısı ihtiva eden bir çok protein, genetik doğru pH Reaksiyon sistein karşı yüksek oranda seçicidir ii) vardır tasarlanmış olabilir, çünkü bu, iii), sulu tamponlar içinde düzgün bir şekilde devam eder ve iv) çok hızlıdır bazı durumlarda, 11 5000 M-1 sn-1 aşan bildirilmiştir sistein içeren proteinlere maleimid ikinci dizi oranı sabiti ile. 12 ilgilenilen protein organik bir küçük (≈% 5-10) miktarı tolere edebilir sağlanan eş-çözücü, hemen hemen her maleimid fonksiyonlu boya, polymer, yüzey veya başka bir protein, protein ile bağlantılı olabilir. Buna ek olarak, maleimitler, yüksek pH diğer nükleofillerle reaksiyona daha yatkındır iyodoasetamid, daha proteinler üzerinde sistein daha özel olarak; ve disülfid değişim 13 önlemek için asidik pH değerinde tutulması gerekir disülfid tabanlı çekimleri daha kararlı. Burada, bir Ru (ll) tabanlı kromofor ve örnek olarak redoks proteini sitokrom c arasındaki reaksiyonu kullanarak tek bir sistein kalıntısı ihtiva eden bir proteine ​​maleimid işlevselleştirilmiş moleküllerin bağlanması için genel bir protokol sunulmuştur. Bu protokol, erişilebilir bir yüzeye sistein kalıntısı ve karşılık gelen maleimid-işlevselleştirilmiş bir hedef içeren, başka proteinler için de geçerlidir, bu bir protein, bir floresan boya, bir kromofor veya sentetik polimer.

<table><tbody><tr><td>sodium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>71496</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>71691</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma-Aldrich</td><td>73575</td><td></td></tr><tr><td>cytochrome &lt;em&gt;c&lt;/em&gt;, from saccaromyces cerevisiae</td><td>Sigma-Aldrich</td><td>C2436</td><td></td></tr><tr><td>dithiothreitol</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>TSKgel SP-5PW</td><td>Sigma-Aldrich</td><td>Tosoh SP-5PW, 07161</td><td>3.3 ml strong cation exchange column</td></tr><tr><td>Amicon Ultra-15&amp;nbsp;</td><td>Merck-Millipore</td><td>UFC900308</td><td>3.5 kDa spin filter</td></tr><tr><td>Slide-A-Lyzer mini dialysis units</td><td>Thermo Scientific</td><td>66333</td><td>3.5 kDa dialysis cassetes</td></tr><tr><td>Ru(II) bisterpyridine maleimide</td><td>Lab made</td><td></td><td>see ref (14)</td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>A3396</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>03609</td><td></td></tr><tr><td>tris(2-carboxyethyl)phosphine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>93284</td><td></td></tr><tr><td>imidazole</td><td>Sigma-Aldrich</td><td>56749</td><td></td></tr><tr><td>nickel acetate</td><td>Sigma-Aldrich</td><td>244066</td><td></td></tr><tr><td>AcroSep IMAC Hypercell column</td><td>Pall</td><td>via VWR: 569-1008</td><td>1 ml IMAC column</td></tr><tr><td>0.2 micron cellulose membrane filter</td><td>Whatman</td><td>Z697958</td><td>47 mm filter for buffers</td></tr><tr><td>0.2 micron PVDF membrane filter</td><td>Merck-Millipore</td><td>SLGV013SL</td><td>syringe filters for proteins</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>84426</td><td>Extremely corrosive! Use caution.</td></tr><tr><td>caffeic acid</td><td>Sigma-Aldrich</td><td>60018</td><td>MALDI matrix</td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td>extremely corrosive! Use caution</td></tr><tr><td>SimplyBlue SafeStain</td><td>Thermo Scientific</td><td>LC6060</td><td>Coomassie blue solution</td></tr><tr><td>NuPAGE Novex 12% Bis-Tris Gel</td><td>Thermo Scientific</td><td>NP0342BOX</td><td>precast protein gels</td></tr><tr><td>SeeBlue Plus2 Pre-stained Protein Standard</td><td>Thermo Scientific</td><td>LC5925</td><td>premade protein ladder</td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Thermo Scientific</td><td>NP0008</td><td>premade gel sample buffer</td></tr><tr><td>NuPAGE Sample Reducing Agent (10x)</td><td>Thermo Scientific</td><td>NP0004</td><td>premade gel reducing agent</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Thermo Scientific</td><td>NP0002</td><td>premade gel running buffer</td></tr><tr><td>Voyager DE STR MALDI reflectron TOF MS</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Acta FPLC</td><td>GE</td><td></td><td>Fast Protein Liquid Chromatography</td></tr><tr><td>Cary 50 Bio Spectrophotometer</td><td>Varian-Agilent</td><td></td><td>UV-Vis</td></tr><tr><td>Milli-Q ultrapure water dispenser</td><td>Merck-Millipore</td><td></td><td>ultrapure water</td></tr><tr><td>Low volume UV-Vis Cuvette</td><td>Hellma</td><td>105-201-15-40</td><td>100 microliter cuvette</td></tr></tbody></table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Biyokonjugatlar sentezi, üç ana yöntem ile teyit edilir: flight kütle spektrometrisi (MALDI-TOF MS), poliakrilamid jel elektroforezi Matris destekli lazer dezorpsiyon iyonizasyon süresi ve ultraviyole-Görünür (UV-Vis) spektroskopisi, Şekil 2&#39;de gösterildiği gibi, 3, 4. Ekteki küçük molekülün kütlesine karşılık gelen bir kütle artışı, ve reaksiyona girmemiş protein eksikliği Ru (II), başarılı bir şekilde ...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Bu protokol, reaktif saflaştırma, reaksiyon koşulları, Bioconjugate saflaştırma ve Bioconjugate karakterizasyonu dahil olmak üzere, bir maleimid proteini ihtiva eden bir sisteinin bioconjugation için gerekli olan önemli adımlar göstermektedir.

Protein Bioconjugatlar sentezi<em&gt; yoluyla</em&gt; Sistein-maleimid Kimya

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Chemical Applications of Statistical Analyses

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38.3K Görüntüleme.  The University of New South Wales.  Bu protokol, reaktif saflaştırma, reaksiyon koşulları, Bioconjugate saflaştırma ve Bioconjugate karakterizasyonu dahil olmak üzere, bir maleimid proteini ihtiva eden bir sisteinin bioconjugation için gerekli olan önemli adımlar göstermektedir. 

Video: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Katı Faz Reçine-tabanlı Metodoloji aracılığıyla Asimetrik Ferrosen Türetilmiş Bio-konjuge Sistemleri Sentetik Metodoloji

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Kimya

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Sistein Kalıntılarının Solvent Erişilebilirlik Analizi<em&gt; Mısır rayado fino virüsü</emÜretilen&gt; Virüs gibi parçacıklar<em&gt; Nicotiana benthamiana</emVLP&#39;ler için Peptidlerin&gt; Bitkiler ve Çapraz bağlama

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

İmmünoloji ve Enfeksiyon

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Thioether/vinil sülfür-gergin helisel peptidler fotoğraf yolu ile indüklenen Thiol-KD/yne Hydrothiolation oluşturmak

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Genetik bir kanonik olmayan Amino asit antikor-uyuşturucu üretimi için kodlama Conjugates bir hızlı Bioorthogonal tepki

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Sentez ve μ-Conotoxin PIIIA izomerler farklı disülfür Connectivities ile yapı belirlenmesi

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Sentez ve Site seçici olarak oluşturulması için Thiol reaktif reaktif Bioconjugation Immunoconjugates modifiye

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

Biyokimya

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bakteri-konak Etkileşimleri karakterize etmek Biyomimetik Malzemeler

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Hepatit E virüs nano tanecikleri kimyasal konjugasyon yöntemlerle yüzey Functionalization

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Biyomühendislik

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Sistein Thiols rekombinant proteinler Vitro siteye özgü Glikozilasyon için hedefleme

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Protein Bioconjugatlar sentezi

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