Name: efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition
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Description: Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

1. 플라스미드 건설 <ol><li> 그의 6 -tag와 대상 항체 유전자 (pBAD-HerFab-WT)을 표현하는 것 발현 플라스미드 (pBAD-HerFab - L177TAG)를 구축하고, 앰버 코돈과 류신-177의 코돈을 대체 (TAG) 27 사용 종래의 부위 특이 적 변이 법. 재료의 표를 참조하십시오. </li><li> 진화의 tRNA 티르와 아미노 아실 tRNA의 합성 (Aars의) 한 쌍의 유전자를 포함하는 또 다른 발현 플라스미드 (pEVOL-AFRS)를 구축합니다. UAAs의 효율적인 통합을 위해 특별히 디자인 된 플라스미드 벡터, pEVOL을 사용합니다. 상세한 정보 및 플라스미드 복제 프로토콜은 이전 보고서에 기재되어있다. (28) </li><li> 시중에서 판매하는 플라스미드 준비 키트를 사용하여 높은 품질의 플라스미드 DNA를 얻습니다. ~ 1.8의 280 분의 260 비율은 정제 된 DNA에 최적입니다. 필요한 경우, DNA의 순도를 확인하는 1 % 아가 로스 겔 전기 영동을 수행한다. </li></ol><ol><li> 전기 <ol><li> 20 μL 대장균 DH10β 균주에 pEVOL-AFRS 28 pBAD-HerFab - L177TAG 각각 1 μL의 DNA를 추가합니다. 피펫을 사용하여 부드럽게 섞는다. 플라스미드를 함께 또는 독립적 인 반응으로 변형 될 수있다. </li><li> 0.1 cm 큐벳를 들어, 25 μF 2.5 kV의에 전기 설정을 설정합니다. </li><li> 충격적인 챔버의 슬라이드에 큐벳을 삽입합니다. 큐벳 챔버 전극에 닿을 때까지 챔버로 슬라이드를 밀어 넣습니다. </li><li> 일렉트로 기계 경고음까지 펄스 버튼을 눌러 한 번 펄스. </li><li> catabolite 억압 (SOC) 배지, 효모 추출물, 10 mM의 염화나트륨, 2.5 mM의 KCl을 (/ V W) 트립 톤, 0.5 % (/ V W), 10 밀리미터의 MgCl 2, 2 %를 함유하는 1 ㎖의 슈퍼 최적 국물, 20 mM의 글루코스를 추가 신속 큐벳 및 테스트 튜브에 혼합물을 옮긴다. 진탕하면서 37 ℃에서 1 시간 동안 세포를 인큐베이션.</li><li> (pEVOL-AFRS 각각 pBAD-HerFab - L177TAG을 선택하기위한 35 μg의 / ML의 클로람페니콜과 100 μg의 / ML의 암피실린) 적절한 항생제를 포함하는 판 한천 원성 국물 (LB)의 확산 형질 전환 된 대장균 세포. 12 시간 동안 37 ° C에서 접시를 품어. </li></ol></li><li> 문화의 준비 <ol><li> 5 ㎖ LB 배지 포함하는 항생제의 단일 변환 식민지의 예방. 진탕 37 ° C에서 12 시간 동안 품어. </li></ol></li></ol> 3. 발현 및 HerFab-L177AF의 정제 <ol><li> HerFab - L177AF의 발현 <ol><li> 200 mL의 LB 배지에 암피실린을 함유하는 일차 배양 (5 ml)에 전송 (100 μg의 / mL) 및 AF (1 mM)을 진탕하면서 37 ℃에서 배양 하였다. 주 : 100 mM의 AF 스톡 용액 (10 ㎖)을 100 mM의 염산 (10 ㎖, 최종 부피)을 206 mg의 AF를 용해시켜 제조 하였다. </li><li> (16 1.6 M 아라비 노스의 2 mL를 넣고문화 로그 상 (OD 550 = 0.8, OD = 광학 밀도)에 도달 mM의 최종 농도). 진탕 30 ° C에서 12 시간 동안 품어. </li><li> 5 분 동안 11,000 XG에 원심 분리하여 수확 세포. 뜨는을 취소하고 -20 ° C에서 펠렛을 동결. </li></ol></li><li> 세포 용해 <ol><li> 30 mM 트리스 (PH 8.0), 1 mM의 EDTA, 20 % 수 크로스, 0.2 ㎎ / ㎖의 리소자임을 함유하는 20 ㎖ 세포질 용해 완충액에서 세포 펠렛을 재현 탁하고, 37 ° C에서 1 시간 동안 혼합물을 배양한다. </li><li> 15 분 동안 4 ℃에서 18,000 × g으로의 세포 용 해물을 원심 분리기. 새로운 튜브에 뜨는을 전송하고 펠렛을 폐기합니다. </li></ol></li><li> 니켈 NTA 친 화성 크로마토 그래피 <ol><li> 각각의 원심 분리기 튜브 (200 mL의 문화 400 μL 수지)에 니켈 NTA 수지 서스펜션을 추가하고 1 시간 동안 4 ° C에서 부드럽게 섞는다. </li><li> 폴리 프로필렌 열로 현탁액을 붓고 5 mL로 세척 버퍼와 수지 세 번 씻어ffer의 50 mM의 NaH 2 PO 4 (PH 8.0), 20 mM의 이미 다졸, 및 300 mM의 NaCl을 함유하는. </li><li> 50 밀리미터에 NaH 2 PO 4 (PH 8.0), 250 mM의 이미 다졸, 및 300 mM의 NaCl을 함유하는 300 μL 용출 완충액 표적 항체를 용출시켰다. </li><li> 브래드 포드 단백질 분석 (29)에 의해 또는 280 nm에서 흡광도를 측정하여 돌연변이 단백질의 농도를 결정합니다. 흡광 계수 (2471 M -1 cm-1)에 대한 AF를 사용하여 단백질 흡광 계수 산출하여 280 nm에서 HerFab-L177AF 대한 흡광 계수 (75,866 M -1 cm-1)를 계산한다. 주 : HerFab의 아미노산 서열은 NCBI 웹 사이트에서 발견 (GI : 783282791 및 GI : 783282792)을 할 수있다. </li></ol></li></ol> 스트레인 승진 아 지드 - 알킨 사이클로을 사용하여 알킨 프로브를 정화 HerFab-L177AF 4. 활용 (SPAAC) <ol><li> (H 2 O에 200 Cy5.5-Azadibenzocyclooctyne (Cy5.5-ADIBO) 20 μL를 추가81, 10 mM의 나트륨 2 HPO 4 (pH 7.0) 및 100mM NaCl을 함유하는 인산염 완충액 HerFab-L177AF 10 μL (10 μM 최종 농도)의 용액에 M, 최종 농도). 참고 : 대안, 다이 플루오르 cyclooctyne (DIFO) 및 시클로 [6.1.0] nonyne (BCN) 파생 상품은 같은 응용 프로그램을 사용할 수 있습니다. </li><li> 스트레인 승진 고리 화 반응은 37 ℃에서 6 시간 동안 진행 할 수 있습니다. 감광성 프로브 (예, Cy5.5)를 사용하는 경우, 알루미늄 박과의 반응 용기 커버. </li><li> 5 단계에 따라 표시 HerFab을 정화. </li></ol> 레이블이 HerFab 5. 정제 <ol><li> 원심 필터 스핀 컬럼에 시료 500 μL를 추가합니다. </li><li> 15 분 동안 4 ° C에서 14,000 XG에 스핀 컬럼을 원심 분리기. </li><li> 폐기 관류 및 전송에 1.5 ㎖의 마이크로 원심 튜브에 스핀 컬럼으로부터 샘플을 정제 하였다. </li><li> 대안 5.1 5.3 단계로서, 정수기를 수행동일한 완충액에 대해 투석하여 기. </li><li> 4 ° C에서 정제 된 라벨 HerFab를 저장합니다. </li></ol> 레이블이 HerFab 6. SDS-PAGE 분석 <ol><li> 106 mM 트리스 · 염산, 141 mM 트리스베이스 (PH 8.5), 2 % LDS, 10 % 글리세롤, 0.51 mM의 EDTA, 0.22 밀리미터의 Serva 블루 G250, 그리고 13 μL에 빨간색 0.175 mM의 페놀을 포함하는 5 μL LDS 단백질 샘플 버퍼를 추가 존재 (+) 또는 부재 HerFab (7.8 μM 또는 0.38 ㎎ / ㎖)을 표지 된 정제 된 (-) 2 μL 디티 오 트레이 톨 (DTT, 100 mM의 최종 농도). 10 분 동안 95 ℃에서 혼합물을 인큐베이션. </li><li> 전기 영동 셀에 4-12% 비스 - 트리스 SDS-PAGE 겔 카세트를 부착하고 버퍼를 실행에 추가합니다. 공액 단백질 샘플 및 사전 스테인드 분자량 마커를로드합니다. 200 V. 35 분간 전기 영동을 수행 참고 : 형광의 사진 표백을 최소화하기 위해 전체 기간 동안 어둠 속에서 전기 영동 셀을 유지합니다. </li><li> 전기 영동 후, 전송형광 젤 스캐너에 젤, 그리고 적절한 파장에서 형광 방출을 검색. Cy5.5 들어 스캐너 소프트웨어에서 Cy5의 모드를 사용한다. </li><li> 상업 단백질 얼룩 젤을 얼룩. </li></ol>

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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.

단백질에 비 천연 아미노산의 유전 결합 단백질 변성에 사용되는 다른 방법에 비해 여러 가지 장점을 갖는다. 중요한 장점 중 1-3 하나는 단백질의 종류는 일반적으로 적용됩니다. 원칙적으로, 표적 단백질 및 단백질의 표적 부위를 선택에는 제한이 없다. 그러나 UAA와 구조적 또는 기능적으로 중요한 잔기의 치환은 목적 단백질의 구조와 기능을 변화 될 수있다. 일반적으로, 용매에 노출되...

대장균에서 P는 -methoxyphenylalanine의 유전 적 결합이보고 된 이후, 한 100 개 이상의 비 천연 아미노산 (UAAs가) 성공적으로 다양한 단백질에 통합되었습니다. 이들 중에서도 UAAs 1-3 bioorthogonal 관능기를 함유하는 아미노산은 광범위하게 연구하고, 최대 비율을 표현하고있다. UAAs에 사용되는 bioorthogonal 기능 그룹은 케톤, 4 지드, 5 알킨, 6 cyclooctyne, 7 테트라, 8 α, β 불포화 아미드, 9 노보 넨, 10 transcyclooctene, 11, 시클로 [6.1.0] -nonyne을 포함한다. 각 작용기는 장점과 단점이 있지만 (11), 아 지드 - 함유 아미노산이 가장 광범위 단백질 접합에 사용되어왔다. P -Azidophenylalanine (AF), 아지도 - 함유 아미노산 중 하나가 용이 가능하며, 그 혼입 efficiency이 우수합니다. 이 아미노산을 함유하는 변이체 단백질은 구리 촉매 고리 화하거나 SPAAC 의해 cyclooctynes ​​알킨과 반응 할 수있다. 12-20 최근 바이오 제약 산업에서 큰 관심을 받고있다. 항체 약물 접합체 (ADC)는 의한 인간 암 (21) 및 치료 대상 치료 능력에 유리한 치료 항체의 클래스 기타 질병. 50 개 이상의 ADC는 임상 시험에서 현재 및 수는 급속히 증가하고있다. ADC 제품 개발에 많은 요인 효과를 최대화하고 부작용을 최소화하기 위해 고려되어야한다. 이러한 요인 중 효율적이고 부위 특이 적 결합 반응은 항체와 공유 결합을 형성하고, 약물은 매우 중요하다. 공액 반응에 원하는 효율 및 특이성은 bioorthogonal에서 작용기와 결합함으로써 달성 될 수있다특이 항체로 혼입되는 비 천연 아미노산. 여기에 22 ~ 26, 우리에 대한 프로토콜을보고 사이트를-구체적으로 통합 항체 단편으로 AF 및 생화학 프로브 변이체 항체 단편 공역.

<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

그들의 구조와 기능을 연구하는 단백질로 화학 프로브를 소개 할 수 현재 많은 화학 도구가 있습니다. 유용한 방법은 유 전적으로 bioorthogonal 관능기를 함유하는 천연 아미노산을 도입하여 단백질 결합이다. 이 보고서는 사이트 별 항체 결합에 대한 상세한 프로토콜을 설명합니다. 프로토콜은 아 지드 - 함유 아미노산의 유전자 도입하고, 변형 촉진 지드 - 사이클로 알킨 (SPAAC)에 의한 접합 반응의 실험 세부 사항을 포함한다. 이 변형 촉진 반응은 생리적 pH와 온도에서 반응 분자의 단순한 혼합으로 진행하고, 구리 (I) 이온 및 구리 킬레이트 배위자와 같은 추가의 시약을 필요로하지 않는다. 따라서,이 방법은 일반적으로 단백질 결합 항체 약물 접합체 (ADC)는 개발에 유용 할 것입니다.

<html> 이 연구에서, 항체 단편은 부위 - 특이 적 단편에 아 지드 - 함유 아미노산을 도입하고, 변형 cyclooctyne로 돌연변이 된 항체 단편을 반응시켜 형광 물질과 결합 (도 1)이었다. HerFab은 AF가 지드 - 함유 아미노산으로 통합되었다하는 대상 항체 단편으로서 선택 하였다. AF로 교체 HerFab 잔사를 선택하려면 HerFab의 X 선 결정 구조 분석 하였다. 잔류 물을 30 ...

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).

효율적이고 사이트 별 스트레인 승진 아 지드 - 알킨 사이클로에 의해 항체 라벨링

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 San Sebastian

Research

JoVE Journal

Biochemistry

20.6K 조회수.  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).

비디오: 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.

역으로 작은 분자 또는 바이오 센서 애플리케이션을위한 다중 구성 요소 구조를 무력화하는 미세 유체 온칩 캡처 - 고리 화 반응

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

연구

화학

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.

를 사용하여 금 나노 입자의 효율적인 방사성 표지를위한 최적화 된 프로토콜<sup&gt; (125)</sup&gt; I-표시 아 지드 보철 그룹

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 클릭 화학을 통해 바이러스성 표면 Chemoselective 변경

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

면역학 및 감염병학

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.

사진-가교 매핑 및 라이브 포유류 세포에서 Bioorthogonal 화학 GPCRs에 프로브 화학의 유전 결합을 최적화

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.

빠른 Bioorthogonal 반응을 통해 변화 항 체 의약품의 세대에 대 한 정식이 아닌 아미노산의 유전자 인코딩

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.

합성 및 Bioconjugation의 Thiol 반응 시 약 사이트에 선택적으로의 창조에 대 한 수정 Immunoconjugates

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 ...

생화학

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.

형광 염료와 항체 라벨은 자기 단백질 A와 단백질 G 비즈를 사용하여

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.

형광 항생제 프로브를 사용한 세균 저항성 시각화

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.

효율적이고 사이트 별 스트레인 승진 아 지드 - 알킨 사이클로에 의해 항체 라벨링

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