Name: efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition
Uploaded: 2016-12-23T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

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>
	<li>Wang, L., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code.">Expanding the genetic code.</a> Angew. Chem. Int. Ed. 44 (1), 34-66 (2004).</li><li>Wu, X., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+at+the+interface+of+chemistry+and+biology.">Synthesis at the interface of chemistry and biology.</a> J. Am. Chem. Soc. 131 (35), 12497-12515 (2009).</li><li>Liu, C. C., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+new+chemistries+to+the+genetic+code.">Adding new chemistries to the genetic code.</a> Annu. Rev. Biochem. 79, 413-444 (2010).</li><li>Wang, L., Zhang, Z., Brock, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+the+keto+functional+groupto+the+genetic+code+of+Escherichia+coli.">Addition of the keto functional groupto the genetic code of Escherichia coli.</a> proc. Natl. Acad. Sci. U.S.A. 100 (1), 56-61 (2003).</li><li>Chin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+p-azido-L-phenylalanine+to+the+genetic+code+of+Escherichia+coli.">Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.</a> J. Am. Chem. Soc. 124 (31), 9026-9027 (2002).</li><li>Deiters, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+incorporation+of+an+alkyne+into+proteins+in+Esshcerichia+coli.">In vivo incorporation of an alkyne into proteins in Esshcerichia coli.</a> Bioorg. Med. Chem. Lett. 15 (5), 1521-1524 (2005).</li><li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+copper-free+click+chemistry.">Genetically encoded copper-free click chemistry.</a> Angew. Chem. Int. Ed. 50 (17), 3878-3881 (2011).</li><li>Seitchik, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+transcyclooctenes.">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with transcyclooctenes.</a> J. Am. Chem. Soc. 134 (6), 2898-2901 (2014).</li><li>Lee, Y. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+acrylamide+fuctionality.">A genetically encoded acrylamide fuctionality.</a> ACS Chem. Biol. 8 (8), 1664-1670 (2013).</li><li>Lang, K., 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=Genetically+encoded+norbornene+directs+site-specific+cellular+protein+labelling+via+a+rapid+bioorthogonal+raction.">Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal raction.</a> Nat Chem. 4 (4), 298-304 (2012).</li><li>Lang, K., 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=Genetic+encoding+of+bicyclononynes+and+trans-cyclooctenes+for+site-specific+protein+labeling+in+vitro+and+in+live+mammalian+cells+via+rapid+fluorogenic+Diels-Alder+reactions.">Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.</a> J. Am. Chem. Soc. 134 (25), 10317-10320 (2012).</li><li>Jewett, J. C., Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Cu-free+click+chemistry+with+readily+synthesized+biarylazacyclooctynones.">Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones.</a> J. Am. Chem. Soc. 132 (11), 3688-3690 (2010).</li><li>Debets, M. F., 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=Azadibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+++2)+cycloaddition.">Azadibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition.</a> Chem. Commun. 46 (1), 97-99 (2010).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mechanism+to+mouse:+a+tale+of+two+bioorthogonal+reactions.">From mechanism to mouse: a tale of two bioorthogonal reactions.</a> Acc. Chem. Res. 44 (9), 666-676 (2011).</li><li>Debets, M. F., 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=Bioconjugation+with+strained+alkenes+and+alkynes.">Bioconjugation with strained alkenes and alkynes.</a> Acc. Chem. Res. 44 (9), 805-815 (2011).</li><li>Mbua, N. E., Guo, J., Wolfert, M. A., Steet, R., Boons, G. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-promoted+alkyne&#8722;azide+cycloadditions+(SPAAC)+reveal+new+features+of+glycoconjugate+biosynthesis.">Strain-promoted alkyne&#8722;azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.</a> ChemBioChem. 12 (12), 1912-1921 (2011).</li><li>Kuzmin, A., Poloukhtine, A., Wolfert, M. A., Popik, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalization+using+catalyst-free+azide-alkyne+cycloaddition.">Surface functionalization using catalyst-free azide-alkyne cycloaddition.</a> Bioconjug. Chem. 21 (11), 2076-2085 (2011).</li><li>Yao, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorophore+targeting+to+cellular+proteins+via+enzymemediated+azide+ligation+and+strain-promoted+cycloaddition.">Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition.</a> J. Am. Chem. Soc. 134 (8), 3720-3728 (2012).</li><li>Hao, Z., Hong, S., Chen, X., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+bioorthogonal+functionalities+into+proteins+in+living+cells.">Introducing bioorthogonal functionalities into proteins in living cells.</a> Acc. Chem. Res. 44 (9), 742-751 (2011).</li><li>Reddington, S. C., Tippmann, E. M., Jones, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residue+choice+defines+efficiency+and+influence+of+bioorthogonal+protein+modification+via+genetically+encoded+strain+promoted+Click+chemistry.">Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.</a> Chem. Commun. 48 (9), 8419-8421 (2012).</li><li>Chari, R. V. J., Miller, M. L., Widdison, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates:+an+emerging+concept+in+cancer+therapy.">Antibody-drug conjugates: an emerging concept in cancer therapy.</a> Angew. Chem. Int. Ed. 53 (15), 3751-4005 (2014).</li><li>Tsao, M., Tian, F., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+staudinger+modification+of+proteins+containing+p-Azidophenylalanine.">Selective staudinger modification of proteins containing p-Azidophenylalanine.</a> ChemBioChem. 6 (12), 2147-2149 (2005).</li><li>Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+and+efficient+method+for+the+site-specific+dual-labeling+of+proteins+for+single+molecule+fluorescence+resonance+energy+transfer.">A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.</a> J. Am. Chem. Soc. 130 (52), 17664-17665 (2008).</li><li>Milles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+strategies+for+single-molecule+protein+fluorescence.">Click strategies for single-molecule protein fluorescence.</a> J. Am. Chem. Soc. 134 (11), 5187-5195 (2012).</li><li>Jang, S., Sachin, K., Lee, h. J., Kim, D. W., Lee, h. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis.">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjug. Chem. 23 (11), 2256-2261 (2012).</li><li>Kazane, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+antibody+multimers+through+peptide+nucleic+acid+conjugation.">Self-assembled antibody multimers through peptide nucleic acid conjugation.</a> J. Am. Chem. Soc. 135 (1), 340-346 (2012).</li><li>Ko, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+site-specific+antibody+labeling+by+strain-promoted+azide-alkyne+cycloaddition.">Efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition.</a> Bull. Korean Chem. Soc. 36 (9), 2352-2354 (2015).</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> J. Mol. Biol. 395 (2), 361-374 (2010).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Cho, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+extracellular+region+of+HER2+alone+and+in+complex+with+the+Herceptin+Fab.">Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.</a> Nature. 421 (6924), 756-760 (2003).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+:+Diverse+chemical+function+from+a+few+good+reactions.">Click chemistry : Diverse chemical function from a few good reactions.</a> Angew. Chem. Int. Ed. 40 (11), 2004-2021 (2001).</li></ol>

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 ol...

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><tbody><tr><td colspan="4">&lt;strong&gt;1. Plasmid Construction&lt;/strong&gt;</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. &lt;em&gt;et al.&lt;/em&gt; Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. &lt;em&gt;BKCS.&lt;/em&gt; &lt;strong&gt;36 (9)&lt;/strong&gt;, 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 &lt;em&gt;E. coli.&lt;/em&gt; &lt;em&gt;J. Mol. Biol.&lt;/em&gt; &lt;strong&gt;395 (2)&lt;/strong&gt;, 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>500&amp;nbsp;g</td></tr><tr><td>Ethidium bromide</td><td>Alfa Aesar</td><td>L07482</td><td>1 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500&amp;nbsp;g</td></tr><tr><td colspan="4">&lt;strong&gt;2. Culture Preparation&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;2.1 Electroporation&lt;/strong&gt;</td></tr><tr><td>Micro pulser</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>0.1&amp;nbsp;cm electrode gap, pkg. of 50</td></tr><tr><td>Ampicillin Sodium</td><td>Wako</td><td>018-10372</td><td>25 g</td></tr><tr><td>Chloramphenicol</td><td>Alfa Aesar</td><td>B20841</td><td>25 g</td></tr><tr><td>Agar</td><td>SAMCHUN</td><td>214230</td><td>500 g</td></tr><tr><td>SOC medium</td><td>Sigma</td><td>S1797</td><td>100 mL</td></tr><tr><td colspan="4">&lt;strong&gt;3. Expression and Purification of HerFab-L177AF&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;3.1 Expression of Herfab-L177AF&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-azido-L-phenylalanine (AF)</td><td>Bachem</td><td>F-3075.0001</td><td>1 g</td></tr><tr><td>L(+)-Arabinose, 99%</td><td>Acros</td><td>104981000</td><td>100 g</td></tr><tr><td>Hydrochloric acid, 35~37%</td><td>SAMCHUN</td><td>H0256</td><td>500 mL</td></tr><tr><td colspan="4">&lt;strong&gt;3.2 Cell Lysis&lt;/strong&gt;</td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99%</td><td>SAMCHUN</td><td>T1351</td><td>500 g</td></tr><tr><td>EDTA disodium salt dihydrate, 99.5%</td><td>SAMCHUN</td><td>E0064</td><td>1 kg</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td>500 g</td></tr><tr><td>Lysozyme</td><td>Siyaku</td><td>126-0671</td><td>1 g</td></tr><tr><td colspan="4">&lt;strong&gt;3.3 Ni-NTA Affinity Chromatography&lt;/strong&gt;</td></tr><tr><td>Ni-NTA resin</td><td>QIAGEN</td><td>30210</td><td>25 mL</td></tr><tr><td>Polypropylene column</td><td>QIAGEN</td><td>34924</td><td>50/pack, 1 mL capacity</td></tr><tr><td>Imidazole, 99%</td><td>SAMCHUN</td><td>I0578</td><td>1 kg</td></tr><tr><td>Sodium phosphate monobasic, 98%</td><td>SAMCHUN</td><td>S0919</td><td>1 kg</td></tr><tr><td>Sodium Chloride, 99%</td><td>SAMCHUN</td><td>S2907</td><td>1 kg</td></tr><tr><td colspan="4">&lt;strong&gt;4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-promoted Azide-alkyne Cycloaddition (SPAAC)&lt;/strong&gt;</td></tr><tr><td>Cy5.5-ADIBO&amp;nbsp;</td><td>FutureChem</td><td>FC-6119</td><td>1 mg</td></tr><tr><td colspan="4">&lt;strong&gt;5. Purification of Labeled HerFab&lt;/strong&gt;</td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>MILLIPORE</td><td>UFC500396</td><td>96/pack, 500 &amp;#956;L capacity</td></tr><tr><td colspan="4">&lt;strong&gt;6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning&lt;/strong&gt;</td></tr><tr><td>1,4-Dithio-DL-threitol, DTT, 99.5%</td><td>Sigma</td><td>10708984001</td><td>10 g</td></tr><tr><td>NuPAGE LDS Sample Buffer, 4x</td><td>Thermofisher</td><td>NP0007</td><td>10 mL</td></tr><tr><td>MES running buffer</td><td>Thermofisher</td><td>NP0002</td><td>500 mL</td></tr><tr><td>Nupage Novex 4-12% SDS PAGE gels</td><td>Thermofisher</td><td>NO0321</td><td>12-well</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Wako</td><td>031-17922</td><td>25 g</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...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

20.6K 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

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Tersi Küçük Moleküller veya Biosensor Uygulamaları için Çok bileşenli Yapılar hareketsiz için microfluidic On-chip Yakalama sikloeklenme Tepki

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Kimya

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Bir Kullanarak Altın Nanopartiküller Verimli Radyoaktif bir Optimize Protokolü<sup&gt; 125</sup&gt; I-etiketli Azid Protez Grubu

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Bioorthogonal tıklayın Kimya ile Viral Yüzeylerin Kemoselektif Modifikasyonu

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

İmmünoloji ve Enfeksiyon

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

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

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

Kimyasal ve genetik eklenmesi en iyi duruma getirme GPCRs fotoğraf-çapraz eşleme ve canlı memeli hücreleri Bioorthogonal kimyada probları

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

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 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

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Floresan Boyalar ile Antikor Etiketleme Manyetik Protein A ve Protein G Boncuk kullanma

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Floresan Antibiyotik Probları Kullanılarak Bakteriyel Direncin Görüntülenmesi

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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

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

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

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

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler