1. Plazmid Yapı <ol><li> Bir His 6 Etiketleme-hedef antikor geni (pBAD-HerFab-WT) ifade edecek bir ifade plazmid (pBAD-HerFab-L177TAG) Construct ve kehribar kodonu ile lösin-177 için kodonu replace (TAG) 27 kullanılarak konvansiyonel saha-yönlendirilmiş mutagenez tekniği. Malzemelerin Tablo bakın. </li><li> Gelişti tRNA Tyr ve aminoasil-tRNA sentetaz (Aars) çifti için genleri içeren başka bir ifade plazmid (pEVOL-AFRS) Construct. UAAS etkin dahil edilmesi için özel olarak tasarlanmış plazmid vektörü, pEVOL kullanın. Ayrıntılı plazmid bilgi ve klonlama protokolleri önceki raporda açıklanmıştır. 28 </li><li> ticari olarak temin edilebilen bir plazmid hazırlama kiti kullanılarak, yüksek kaliteli plazmid DNA elde. ~ 1.8 260/280 oranı saflaştırılmış DNA için en uygunudur. Gerekirse DNA saflığını kontrol etmek için% 1 agaroz jel elektroforez gerçekleştirin. </li></ol><ol><li> elektroporasyon <ol><li> 20 uL E. coli DH10β suşuna pEVOL-AFRS 28 ve pBAD-HerFab-L177TAG her 1 ul DNA ekleyin. bir pipet kullanılarak, yavaşça karıştırın. plazmidler birlikte ya da bağımsız reaksiyonlarda transforme edilebilir. </li><li> 0.1 cm küvetler için, 25 iF ve 2.5 kV elektroporasyon ayarlarını. </li><li> şok odasının slayt içine küvet yerleştirin. Küvet odası elektrotlar ile sıkı temas edene kadar odasına slayt itin. </li><li> elektroporasyon makinesi bip sesi gelene kadar darbe düğmesine basarak bir kez Pulse. </li><li> Katabolik baskı (SOC) ortamı maya ekstresi, 10 mM NaCI, 2.5 mM KCI (ağ / hac) trıpton,% 0.5 (w / v), 10 mM MgCI2,% 2 ihtiva eden 1 mL Süper uygun suyu, ve 20 mM glukoz ekleme hızlı küvete ve bir test tüpüne karışımı aktarın. çalkalanarak 37 ° C&#39;de 1 saat süreyle inkübe hücreleri.</li><li> (PEVOL-AFRS sırasıyla pBAD-HerFab-L177TAG, seçilmesi için 35 ug / ml kloramfenikol ve 100 ug / ml ampisilin) uygun antibiyotikleri içeren agar plakaları lizojeni etsuyu (LB) üzerine yayıldı transforme E. coli hücreleri. 12 saat boyunca 37 ° C&#39;de inkübe edin. </li></ol></li><li> Kültür hazırlama <ol><li> 5 ml LB ortamı içeren antibiyotiklere tek bir dönüştürülmüş koloni inoküle. çalkalanarak 37 ° C&#39;de 12 saat boyunca inkübe edilir. </li></ol></li></ol> 3. Ekspresyon ve HerFab-L177AF saflaştırılması <ol><li> HerFab-L177AF sentezlenmesi <ol><li> 200 ml LB ortamı ihtiva ampisilin içine primer kültürünün (5 mi) transfer (100 ug / ml) ve AF (1 mM), çalkalanarak 37 ° C&#39;de kuluçkalandı. Not: 100 mm AF stok çözeltisi (10 mL), 100 mM hidroklorik asit (10 mL, nihai hacim) 206 mg AF çözülmesiyle hazırlanmıştır. </li><li> (16 1.6 M arabinoz 2 mL ekleyinKültür log-faz (OD 550 = 0.8 OD = optik yoğunluk) ulaşır mM son konsantrasyon). çalkalama ile 30 ° C&#39;de 12 saat boyunca inkübe edilir. </li><li> 5 dakika boyunca 11.000 x g&#39;de santrifüjleme ile hasat hücreleri. Süpernatantı atın ve -20 ° C&#39;de pelet dondurmak. </li></ol></li><li> Hücre liziz <ol><li> 30 mM Tris (pH 8.0), 1 mM EDTA,% 20 sakroz ve 0.2 mg / ml lisozim içeren 20 mi periplazmik liziz tamponunda yeniden süspanse hücre pelletleri, 37 ° C&#39;de 1 saat süreyle inkübe karışımı. </li><li> 15 dakika boyunca 4 ° C&#39;de 18.000 x g&#39;de hücre lizat santrifüjleyin. yeni bir tüp süpernatant aktarın ve pelet atın. </li></ol></li><li> Ni-NTA afinite kromatografisi <ol><li> Her bir santrifüj tüpüne (200 ml kültür, 400 uL reçine) Ni-NTA reçine süspansiyonu ekleyin ve 1 saat boyunca 4 ° C&#39;de nazik bir şekilde karıştırılır. </li><li> polipropilen sütuna süspansiyon dökün ve 5 ml yıkama met ile reçineyi üç defa yıkamakffer 50 mM NaH 2 PO 4 (pH 8.0), 20 mM imidazol, 300 mM NaCl içeren. </li><li> 50 mM NaH 2 PO 4 (pH 8.0), 250 mM imidazol, 300 mM NaCI ihtiva eden 300 uL yıkama tamponu ile hedef antikor yıkayın. </li><li> Bradford protein deneyi 29 ya da 280 nm&#39;de absorbans ölçülerek mutan protein konsantrasyonunu belirleyin. Sönüm katsayısı (2,471 M -1 cm -1) AF kullanarak protein söndürme katsayısı hesap makinesi tarafından 280 nm&#39;de HerFab-L177AF için sönüm katsayısı (75866 M -1 cm -1) hesaplayın. Not: HerFab amino asit sekans, NCBI web sitesinde bulunabilir (Gl: 783282791 ve GI: 783.282.792) olabilir. </li></ol></li></ol> Gerilme-terfi Azid-alkin Siklokatılma kullanma Alkin Sondalar ile Saf HerFab-L177AF 4. Konjugasyon (SPAAC) <ol><li> (H 2 O 200 Cy5.5-Azadibenzocyclooctyne (Cy5.5-ADIBO) 20 mcL ekleyin81; 10 mM Na-2 HPO 4 (pH 7.0) ve 100 mM NaCl ihtiva eden fosfat tamponu içinde HerFab-L177AF 10 uL (10 uM son konsantrasyon) solüsyonuna M nihai konsantrasyon). NOT: alternatifler, diflorine sikloostin (Difo) ve bisiklo gibi [6.1.0] nonyne (BCN) türevleri ayrıca aynı uygulama için kullanılabilir. </li><li> suşa teşvik siklo-ilave reaksiyonu, 37 ° C&#39;de 6 saat devam etmesine izin verir. Işığa duyarlı bir prob (örneğin, Cy5.5) kullanılırsa, alüminyum folyo ile reaksiyon bölmesi kapağı. </li><li> Adım 5&#39;e göre etiketlenmiş HerFab arındırın. </li></ol> Etiketli HerFab 5. saflaştırılması <ol><li> Bir santrifüj filtre döndürme kolonuna numune 500 ul ekle. </li><li> 15 dakika boyunca 4 ° C&#39;de 14,000 x g&#39;de spin kolon santrifüjleyin. </li><li> Sil akış ve transfer 1.5 ml mikrosantrifüj tüpüne spin kolon numuneyi saflaştırılmıştır. </li><li> Alternatif bir 5,1- 5.3 adım olarak, arıtma için yerineaynı tampona karşı diyaliz ile tion. </li><li> 4 ° C&#39;de arıtılmış, etiketli HerFab saklayın. </li></ol> Etiketli HerFab 6. SDS-PAGE analizi <ol><li> 106 mM Tris-HCI, 141 mM Tris bazı (pH 8.5),% 2 LDS,% 10 gliserol, 0.51 mM EDTA, 0.22 mM SERVA Mavi G250 ve 13 uL kırmızı 0.175 mM fenol ihtiva eden 5 uL LDS proteini örnek tamponu ekleme varlığı (+) veya yokluğunda HerFab (7.8 uM veya 0.38 mg / ml) olarak tanımlanan saflaştırılmış (-) 2 uL ditiotreitol (DTT, 100 mM nihai konsantrasyon). 10 dakika boyunca 95 ° C&#39;de inkübe karışımı. </li><li> Elektroforez hücreye% 4-12 Bis-Tris, SDS-PAGE jeli kaseti takın ve çalışan tampon ekleyin. birleşik protein örnekleri ve Önceden boyanmış molekül ağırlığı işaretleyici yükleyin. 200 V 35 dakika boyunca jel elektroforezi gerçekleştirin Not: fluorofor fotoğraf beyazlatma en aza indirmek için tüm dönem boyunca karanlıkta elektroforez hücresi tutun. </li><li> Elektroforezisten sonra, aktarmaBir floresan jel tarayıcıya jel ve uygun dalga boyunda floresan emisyonu için tarayın. Cy5.5 için tarayıcı yazılımı Cy5 modunu kullanın. </li><li> ticari bir protein boyası ile jel Leke. </li></ol>

<ol>
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The authors have nothing to disclose.

proteinlerin içine Doğal olmayan amino asitlerin genetik dahil protein modifikasyonu için kullanılan diğer yöntemlere göre bazı avantajları vardır. Önemli avantajlar 1-3 bir proteinin her türlü genel uygulanabilmesidir. Prensip olarak, bir hedef proteini ve protein, bir hedef sitesi seçiminde herhangi bir sınırlama yoktur. Bununla birlikte, UAA ile yapısal ve fonksiyonel olarak önemli kalıntısının yerine, hedef proteinin yapısını ve fonksiyonunu değiştirerek neden olabilir. Genel olarak, çözücü maruz kalan ve diğer artıkları ile etkileşmeyen tortuları UAAS dahil edilmesi için tercih edilmektedir. Bu nedenle, yüksek çözünürlüklü bir kristal yapısından yapısal bilgi UAA dahil edilmek için uygun sitesi seçmek için kullanılır. 30 UAAS dahil teknik olarak kolaydır ve basit bir mutagenez gerektirdiğinden, birden fazla bölgede kolayca hedef proteinin arzu edilen bir işlev için en uygun bir tortu seçmek için taranabilir. &lt;/ P&gt; Bu protokol, AF genetik bir antikor fragmanı içine dahil edilir, ve mutant parçası SPAAC kullanarak site-spesifik olarak bir florofor ile işaretlenmiş olan. Bu yöntemin konjugasyon verimi önceki rapora kıyasla önemli bir gelişmedir istenmeyen reaksiyon olmadan kantitatiftir. 25 Bu, optimal site seçimi ve reaksiyon süresi bir artış dikkat yapısal analizi ile elde edilmiştir. Bu nedenle, UAA dahil edilmek üzere alana seçimi, hızlı ve nicel konjugasyonu için kritik önem taşır. Buna ek olarak, etkili bir UAA dahil sistemlerinin kullanımı (örneğin, pEvol-AFRS) aynı zamanda, protein verimi ve dahil verimliliği için önemlidir. 28 Protein konjugasyon için genetik dahil yöntemi ile protein içine dahil diğer UAA&#39;lar antikor konjugasyon için kullanılabilir. Reaksiyon oranı cinsinden 4-11, bakır katalize siklo-ilave reaksiyonu,gibi tetrazinler 8 ve gergin alkenler ve alkinleri kullanılarak ters elektron talep Diels-Alder reaksiyonu gibi, 7, bu çalışmada kullanılan SPAAC daha iyi olacaktır. Bununla birlikte, bakır ile katalize edilen siklo-ilave reaksiyonu, genellikle, sentetik olarak zor, ve kantitatif olarak bağlanması için yeteri kadar sabit değildir, bakır (I) &#39;in iyon ve ligandları, 31 ve Diels-Alder reaksiyonu için amino asit gerektirmektedir. Keton-hidroksiamin yoğunlaştırma 4, aynı zamanda, aynı amaç için kullanılabilir. Bununla birlikte, orta seviyede asidik koşullar gerektirir, ve reaksiyon hızı SPAAC daha yavaştır. örneğin, ticari veya sentetik, doğal olmayan amino asitler erişilebilirliği reaksiyon oranı, biyo-ortogonal, reaksiyon koşulları biyouyumluluk ve doğal olmayan amino asitlerin dahil edilmesi, verimliliği, genetik olarak SPAAC ve kullanma yöntemi gibi faktörler göz önüne alındığında AF modifiye geliştirmek için yararlı olacaktır Incorporated tedavi böyle bir ADC gibi proteinler, yanı sıra, genel gelinceProtein etiketleme.

Escherichia coli içinde bir p -methoxyphenylalanine genetik dahil belirtildikten sonra, 1 100&#39;den doğal olmayan amino asitler (UAA&#39;lar) başarılı bir şekilde çeşitli proteinlerin dahil edilmiştir. Bu UAAS arasında 1-3, bioorthogonal fonksiyonel grupları içeren amino asitler yoğun çalışılan ve büyük oranını temsil edilmiştir. UAAS kullanılan bioorthogonal fonksiyonel gruplar, keton, 4 azit 5 alkin, 6 sikloostin, 7 tetrazin, 8 α, β-doymamış bir amid, 9 norbonen, 10 transcyclooctene, 11 ve bisiklo [6.1.0] -nonyne bulunmaktadır. Her fonksiyonel grubun avantajları ve dezavantajları vardır 11 da, azid ihtiva eden amino asitler en yaygın protein konjugasyon için kullanılmıştır. p -Azidophenylalanine (AF), azido-içeren amino asitler bir, hali hazırda mevcuttur, ve dahil efficiency mükemmel. Bu amino asidi içeren mutant proteinler bakır katalitik siklo veya SPAAC göre cyclooctynes ​​sahip alkinler ile reaksiyona sokulabilir. 12-20 Son zamanlarda, biopharmaceuticals ilaç endüstrisinde büyük ilgi çekmiştir. Antikor-ilaç konjugatı (ADC) bağlı insan kanserlerinin 21 ve tedavisi için hedeflenen bir tedavi için kabiliyetleri de avantajlı terapötik bir antikor sınıfı diğer hastalıklar. 50&#39;den fazla ADC klinik çalışmalarda şu anda, ve sayısı hızla artmaktadır. ADC&#39;ler geliştirilmesinde birçok faktör etkinliğini en üst düzeye çıkarmak ve yan etkileri en aza indirmek için dikkate alınması gerekir. Bu faktörler arasında, etkin ve yere-özgü konjugasyonu reaksiyonu, bir antikor arasında bir kovalent bağ oluşturmak üzere bir ilacın önemlidir. Konjugasyon reaksiyonu istenen etkinlik ve spesifikliği, bir bir bioorthogonal fonksiyonel grup ile konjugasyon ile elde edilebilirözellikle, bir antikor içine dahil edilir, doğal olmayan bir amino asit. Burada 22-26, biz bir protokol rapor sitenin spesifik dahil bir antikor fragmanına AF biyokimyasal prob mutant antikor fragmanı konjuge.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. plasmid Construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pBAD_HerFab_L177TAG</td>
			<td></td>
			<td></td>
			<td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td>
		</tr>
		<tr>
			<td>plasmid pEvol-AFRS</td>
			<td></td>
			<td></td>
			<td>Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)</td>
		</tr>
		<tr>
			<td>DH10B</td>
			<td>Invitrogen</td>
			<td>C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr>
			<td>Plasmid Mini-prep kit</td>
			<td>Nucleogen</td>
			<td>5112</td>
			<td>200/pack</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Intron biotechnology</td>
			<td>32034</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Alfa Aesar</td>
			<td>L07482</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>2. Culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1) Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser&#160;</td>
			<td>BIO-RAD</td>
			<td>165-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser cuvette</td>
			<td>BIO-RAD</td>
			<td>165-2089</td>
			<td>0.1cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Wako</td>
			<td>018-10372</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Alfa Aesar</td>
			<td>B20841</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>SAMCHUN</td>
			<td>214230</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Sigma</td>
			<td>S1797</td>
			<td>100ML</td>
		</tr>
		<tr>
			<td>3. Expression and purification of HerFab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.1 Expression of Herfab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine (AF)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose, 99%</td>
			<td>Acros</td>
			<td>104981000</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 35~37%</td>
			<td>SAMCHUN</td>
			<td>H0256</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>3.2 Cell lysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane, 99%</td>
			<td>SAMCHUN</td>
			<td>T1351</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate, 99.5%</td>
			<td>SAMCHUN</td>
			<td>E0064</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Siyaku</td>
			<td>126-0671</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>3.3 Ni-NTA Affinity Chromatography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td>25ml</td>
		</tr>
		<tr>
			<td>Polypropylene column</td>
			<td>QIAGEN</td>
			<td>34924</td>
			<td>50/pack, 1ml capacity</td>
		</tr>
		<tr>
			<td>Imidazole, 99%</td>
			<td>SAMCHUN</td>
			<td>I0578</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic, 98%</td>
			<td>SAMCHUN</td>
			<td>S0919</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium Chloride, 99%</td>
			<td>SAMCHUN</td>
			<td>S2907</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5.5-ADIBO&#160;</td>
			<td>FutureChem</td>
			<td>FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr>
			<td>5. Purification of Labeled HerFab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td>MILLIPORE</td>
			<td>UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr>
			<td>6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td>Sigma</td>
			<td>10708984001</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer, 4X</td>
			<td>Thermofisher</td>
			<td>NP0007</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>MES running buffer</td>
			<td>Thermofisher</td>
			<td>NP0002</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>Nupage Novex 4-12% SDS PAGE gels</td>
			<td>Thermofisher</td>
			<td>NO0321</td>
			<td>12well</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Typhoon 9210 variable mode imager</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition

kendi yapısını ve fonksiyonunu incelemek için proteinlere kimyasal sondaları tanıtmak için mevcut şu anda birçok kimyasal araç vardır. Faydalı bir yöntem, genetik olarak bioorthogonal fonksiyonel grubu ihtiva eden bir doğal olmayan amino asidin sokulması protein konjugasyon olup. Bu rapor, site-spesifik bir antikor konjugasyonu için ayrıntılı bir protokol açıklamaktadır. protokol bir azid içeren amino asitin genetik dahil, ve zorlanma-terfi azid-alkin siklo (SPAAC) tarafından konjugasyon reaksiyonu için deneysel ayrıntıları içerir. Bu soy-destekli Reaksiyon fizyolojik pH ve sıcaklıkta reaksiyona moleküllerin basit karıştırma ile ilerler ve bakır (I) &#39;iyonları ve bakır kenetleme ligandlar olarak ilave reaktifler gerektirmez. Bu nedenle, bu yöntem, genel protein konjugasyon ve antikor ilaç konjugatları (ADC) geliştirilmesi için yararlı olabilecektir.

<html> Bu çalışmada, bir antikor parçası örneğin bir bölgeye özel fragmanına bir azid ihtiva eden amino asit içeren ve gergin sikloostin mutant antikor fragmanının reaksiyona sokulmasıyla bir florofor ile konjuge (Şekil 1). HerFab AF bir azid ihtiva eden amino asit dahil edildiği içine hedef antikor fragmanı olarak seçilmiştir. AF değiştirilmesi için HerFab kalıntı seçmek için HerFab X-ışını kristal yapısı analiz edilmiştir. Kalıntının 30 önemli şartlar etkin konjugasyon reaksiyonu için bağlanma afinitesinde azalmaya ve çözücü ulaşmak en aza indirmek için, antikor bağlanma yeri yeterli mesafe bulunmaktadır. Kalıntı iyi antikor dışına maruz kalan ve antikor bağlanma yeri uzak iki immünoglobulin alanlarının arayüzü, yakın bulunması nedeniyle pozisyon 177 de lösin iyi bir aday oldu. içeriği &quot;fo: keep-together.within Sayfa =&quot; 1 &quot;&gt; Başlangıçta, His6 Etiketleme-bir C-terminal ile birlikte HerFab için ekspresyon plazmidi inşa edildi ve kehribar rengi bir kodon sitesi AF dahil edilmesi için pozisyon 177 tanıtıldı -directed mutagenez. AF içeren mutant HerFab birlikte ifade gelişti tRNA, Tyr ve aminoasil-tRNA sintetaz çift 1mM AF mevcudiyetinde ifade edildi. 27 Ni-NTA afinite saflaştırma mevcudiyetinde ifade mutant proteinler için uygulandı ve AF ve aşağıdaki SDS-PAGE analizinin olmaması tam uzunlukta antikor fragmanı AF mevcudiyetinde sadece elde edildiği (Şekil 2). Daha sonra, AF içeren mutant antikor parçası Cy5.5 ile konjügasyon reaksiyonu için değerlendirildi mevcudiyetinde B-bağlı aza-dibenzocyclooctyne türevi (Cy5.5-ADIBO). antikor fragmanı ve Cy5.5-ADIBO 6 saat bir fosfat tamponu içinde reaksiyona sokuldu ve reaksiyon karışımı, SDS-PAGE ile analiz edilmiştir (+) ve yokluk<html> (-) DTT (Şekil 3a). 25. Bir vahşi tip bir antikor fragmanı ile reaksiyonu, aynı zamanda gerçekleştirilen bir kontrol olarak analiz edildi. konjugasyon vahşi tip fragmanı ile reaksiyonda gözlenirken floresans görüntüleri net, Cy5.5-ADIBO mutant antikor fragmanının konjugasyon göstermiştir. Elektron püskürtme iyonizasyon kütle spektrometrisi (ESI-MS) analizi hiç bir bulgulanabilir birleşmemiş fragmanının (Şekil 3b) kantitatif konjugasyon göstermiştir. Genel olarak, bu sonuçlar, AF içeren mutant HerFab ek reaktif olmayan etkin ve uygun şekilde SPAAC tarafından gergin cyclooctynes ​​ile konjuge olabilir gösterdi. <img alt="Şekil 1" src="/files/ftp_upload/54922/54922fig1.jpg" /> Şekil 1: SPAAC site-spesifik antikor etiketleme şematik gösterimi.&quot;Blank&gt; Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="şekil 2" src="/files/ftp_upload/54922/54922fig2.jpg" /> Şekil 2: gelişmiş tRNA / AARS çiftinin mevcudiyetinde pozisyon 177 (L177) AF içeren mutant HerFab ekspresyonu. gelişti tRNA / AFRS çifti ve 1 mM AF içeren LB ortamında HerFab-L177AF sentezlenmesi. (-) Ile saflaştınlmış numuneler mevcudiyetinde (+) veya yokluğunda SDS-PAGE ile analiz edildi DTT ve jel, ticari bir protein lekesi ile boyandı. Ko, W. ve arkadaşları uyarlanmıştır Şekil. 27. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54922/54922fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/54922/54922fig3.jpg" /> Şekil 3: Konjugasyon tepkiCy5.5-ADIBO ile HerFab-L177AF iyon. Cy5.5-ADIBO vahşi tip HerFab ve HerFab-L177AF konjugasyonu reaksiyonu (a), SDS-PAGE analizi. (-) Reaksiyon karışımları varlığında (+) ve yokluğunda analiz edildi DTT ve protein bantları, ticari bir protein leke (sol) ve floresan görüntüleme (sağ) ile görselleştirilmiştir. (B) ESI-MS Cy5.5-ADIBO (sağ) ile etiketlenmiş HerFab-L177AF (sol) ve HerFab-L177AF analizleri: HerFab- L177AF ve konjuge antikor = 1,190 Da arasında beklenen kütle farkı kütle farkı = 1,190 Da gözlenen . Ko, W. ve arkadaşları uyarlanmıştır Şekil. 27. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54922/54922fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Verimli ve Siteye özgü Gerilme-terfi Azid-alkin Siklokatılma tarafından Antikor Etiketleme

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

efficient-site-specific-antibody-labeling-strain-promoted-azide

Nanyang Technological University

Research

JoVE Journal

Biochemistry

20.5K Görüntüleme.  Sogang University. Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Video: Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Canlı hücre fotoafinite Etiketleme tarafından Yerli Hücresel Ortamında Küçük Molekül bağlayıcı proteinlerin belirlenmesi

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Biyokimya

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Döner disülfür Çapraz bağlı misellerin Üretimi için basit ve etkin Yaklaşım

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Laboratuar ölçekli üretim ve terapötik bir antikor ile saflaştırılması

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

Geliştirilmiş Numune Kombine Öncül İzotop Etiketleme ve Isobaric Etiketlemeyi Kullanma Dokuların çoğullama (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Histon Antikor Özgüllüğünün Peptit Mikroarraylerle Analizi

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Derin Proteom izobarik etiketleme, kapsamlı sıvı Kromatografi, kütle spektrometresi ve yazılım destekli miktar tarafından profil oluşturma

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Verimli Refolding ve arıtma Chemoreceptor Ligand bağlayıcı etki için yöntemi

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Yalıtım Glomeruli ve Glomerular hücre yüzey proteinleri Vivo etiketleme

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Proteom çapında Quantification maddelerinin homojenliği tek molekül düzeyinde etiketlenmesi

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Bir Kombinatoryal Tek Etki Alanı Antikor Kütüphanesi adım wise Faj Seçimi ile Son Derece Spesifik Kimyasal Kaynaklı Protein Dimerization Sistemleri Oluşturma