Effektiv og site-specifikt antistof Mærkning af stamme-forfremmet azid-alkyn Cycloaddition
Summary
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).
Abstract
Der er i øjeblikket mange kemiske værktøjer til rådighed til at indføre kemiske sonder i proteiner til at studere deres struktur og funktion. En nyttig metode er protein konjugering ved genetisk indføre en unaturlig aminosyre indeholdende en bioorthogonal funktionel gruppe. Denne rapport beskriver en detaljeret protokol til site-specifikt antistof konjugation. Protokollen omfatter eksperimentelle detaljer til genetisk inkorporering af et azid-indeholdende aminosyre, og konjugeringsreaktionen ved stamme-fremmet azid-alkyn cycloaddition (SPAAC). Denne stamme-fremmet reaktionen forløber ved simpel blanding af de reagerende molekyler ved fysiologisk pH og temperatur, og kræver ikke yderligere reagenser såsom kobber (I) ioner og kobber-chelaterende ligander. Derfor ville denne fremgangsmåde være nyttig til generel protein konjugering og udvikling af antistof-lægemiddelkonjugater (ADC'er).
Introduction
Da den genetiske inkorporering af p -methoxyphenylalanine i Escherichia coli blev rapporteret, har 1 mere end 100 unaturlige aminosyrer (UAAs) blevet indarbejdet i forskellige proteiner. 1-3 Blandt disse UAAs har de aminosyrer indeholdende bioorthogonal funktionelle grupper blevet grundigt undersøgt og repræsenterer den største andel. De bioorthogonal funktionelle grupper anvendes i UAAs omfatter keton, 4 azid, 5 alkyn, 6 cyclooctyne, 7 tetrazin, 8 α, β-umættede amid, 9 norbonene, 10 transcyclooctene, 11 og bicyclo [6.1.0] -nonyne. 11 Selv om hver funktionel gruppe har sine fordele og ulemper, har azidet-holdige aminosyrer er mest udbredt anvendt for protein konjugering. p -Azidophenylalanine (AF), en af azido-holdige aminosyrer, er let tilgængelige, og dets inkorporering effekiency er fremragende. Mutantproteiner indeholdende denne aminosyre kan omsættes med alkyner af kobber-katalyseret cycloaddition eller med cyclooctynes efter SPAAC. 12-20
For nylig er biopharmaceuticals tiltrukket stor opmærksomhed i den farmaceutiske industri. Den Antistoflægemiddelkonjugat (ADC) er en klasse af terapeutiske antistoffer, der er fordelagtige på grund af deres evne til målrettet terapi til behandling af humane cancere 21 og andre sygdomme. Mere end 50 ADCs er i øjeblikket i kliniske forsøg, og antallet er hastigt stigende. I udvikling af ADC'ere, skal overvejes for at maksimere effektiviteten og minimere bivirkningerne af mange faktorer. Blandt disse faktorer, at en effektiv og stedspecifik konjugering reaktion danner en kovalent binding mellem et antistof og et lægemiddel er kritisk. Den ønskede effektivitet og specificitet i konjugeringsreaktionen kan opnås ved konjugation med en bioorthogonal funktionel gruppe i enunaturlig aminosyre, som er specifikt inkorporeret i et antistof. 22-26 Her rapporterer vi en protokol til site-specifikt indarbejde AF ind et antistoffragment og konjugere det mutante antistoffragment med en biokemisk probe.
Protocol
Representative Results
Discussion
Den genetiske inkorporering af unaturlige aminosyrer i proteiner har adskillige fordele frem for andre metoder, der anvendes for protein modifikation. 1-3 En af de vigtige fordele er dens generelle anvendelighed til nogen form for protein. I princippet er der ingen begrænsning i at vælge et målprotein og et målområde af proteinet. udskiftning af et strukturelt eller funktionelt vigtige rest med en ULA, kan dog resultere i at ændre strukturen og funktionen af målproteinet. Generelt er rester, der …
Declarações
The authors have nothing to disclose.
Materials
| 1. plasmid Construction | |||
| plasmid pBAD_HerFab_L177TAG | 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) | ||
| plasmid pEvol-AFRS | 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) | ||
| DH10B | Invitrogen | C6400-03 | Expression Host |
| Plasmid Mini-prep kit | Nucleogen | 5112 | 200/pack |
| Agarose | Intron biotechnology | 32034 | 500g |
| Ethidium bromide | Alfa Aesar | L07482 | 1g |
| LB Broth | BD Difco | 244620 | 500g |
| 2. Culture preparation | |||
| 2.1) Electroporation | |||
| Micro pulser | BIO-RAD | 165-2100 | |
| Micro pulser cuvette | BIO-RAD | 165-2089 | 0.1cm electrode gap, pkg. of 50 |
| Ampicillin Sodium | Wako | 018-10372 | 25g |
| Chloramphenicol | Alfa Aesar | B20841 | 25g |
| Agar | SAMCHUN | 214230 | 500g |
| SOC medium | Sigma | S1797 | 100ML |
| 3. Expression and purification of HerFab-L177AF | |||
| 3.1 Expression of Herfab-L177AF | |||
| p-azido-L-phenylalanine (AF) | Bachem | F-3075.0001 | 1g |
| L(+)-Arabinose, 99% | Acros | 104981000 | 100g |
| Hydrochloric acid, 35~37% | SAMCHUN | H0256 | 500ml |
| 3.2 Cell lysis | |||
| Tris(hydroxymethyl)aminomethane, 99% | SAMCHUN | T1351 | 500g |
| EDTA disodium salt dihydrate, 99.5% | SAMCHUN | E0064 | 1kg |
| Sucrose | Sigma | S9378 | 500g |
| Lysozyme | Siyaku | 126-0671 | 1g |
| 3.3 Ni-NTA Affinity Chromatography | |||
| Ni-NTA resin | QIAGEN | 30210 | 25ml |
| Polypropylene column | QIAGEN | 34924 | 50/pack, 1ml capacity |
| Imidazole, 99% | SAMCHUN | I0578 | 1kg |
| Sodium phosphate monobasic, 98% | SAMCHUN | S0919 | 1kg |
| Sodium Chloride, 99% | SAMCHUN | S2907 | 1kg |
| 4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) | |||
| Cy5.5-ADIBO | FutureChem | FC-6119 | 1mg |
| 5. Purification of Labeled HerFab | |||
| Amicon Ultra 0.5 mL Centrifugal Filters | MILLIPORE | UFC500396 | 96/pack, 500ul capacity |
| 6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning | |||
| 1,4-Dithio-DL-threitol, DTT, 99.5 % | Sigma | 10708984001 | 10g |
| NuPAGE LDS Sample Buffer, 4X | Thermofisher | NP0007 | 10ml |
| MES running buffer | Thermofisher | NP0002 | 500ml |
| Nupage Novex 4-12% SDS PAGE gels | Thermofisher | NO0321 | 12well |
| Coomassie Brilliant Blue R-250 | Wako | 031-17922 | 25g |
| Typhoon 9210 variable mode imager | Amersham Biosciences |
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