Syntese af Protein biokonjugater<em> via</em> Cystein-maleimid Kemi
Summary
Denne protokol beskriver de vigtige skridt, der er nødvendige for bioconjugation af en cystein indeholder protein til en maleimid, herunder reagens rensning, reaktionsbetingelser, biokonjugat rensning og biokonjugat karakterisering.
Abstract
The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine – maleimide coupling of proteins to other proteins, dyes, drugs or polymers.
Introduction
Bioconjugation indebærer kovalent binding ene biomolekyle med hinanden eller med en syntetisk molekyle, såsom et farvestof, lægemiddel eller en polymer. Protein bioconjugation metoder nu flittigt brugt i mange kemi, biologi og nanoteknologi forskergrupper med ansøgninger fra fluorescerende farvestof mærkning 1,2, hvilket gør af protein (antistof) -prodrugs 3 (antistof medikamentkonjugater – ADC'er) syntese af protein dimerer 4,5 , igennem til selvsamlende protein-polymer-hybrider 6,7 anvendes i nanomedicin 8 og systemer kemi 9.
Specificitet af kemien, der anvendes til bioconjugation, mens ikke altid kritisk, er af største vigtighed for de fleste funktionelt protein biokonjugater, således at ikke forstyrre det aktive sted i målproteinet. Den ideelle bioconjugation reaktion skal opfylde flere kriterier, herunder: i) rettet mod sjældne eller unikke steder på proteinet af interesse,ii) være selektiv mod dette mål, iii) fortsætte under ikke-denaturerende betingelser for at undgå protein udfoldning og iv) være højtydende som målproteinet er sædvanligvis kun tilgængelige på sub-millimolær koncentration. Den maleimid – cystein Michael Ud kommer tæt på at opfylde alle disse kriterier, og har derfor længe hævdet en særlig status inden for biokonjugat kemi 10. Dette er fordi i) mange proteiner, der kun indeholder én cysteinrest på deres overflade kan gensplejses der, ii) på den korrekte pH reaktionen er stærkt selektiv dybt cystein, iii) det forløber glat i vandige puffere og iv) det er meget hurtigt med den anden ordens hastighedskonstanten af maleimider til cysteinholdige proteiner rapporteret at overstige 5.000 M -1 sek-1 i nogle tilfælde 11. Forudsat proteinet af interesse kan tolerere en lille (≈ 5-10%) mængden af organisk co-opløsningsmiddel 12, næsten enhver maleimid-funktionaliseret farvestof, polymer, overflade eller et andet protein kan bindes til proteiner. Desuden maleimider er mere specifikke for cysteiner på proteiner end iodacetamider, som er mere tilbøjelige til at reagere med andre nukleofiler ved forhøjet pH; og mere stabile end disulfid-baserede bøjninger, som skal holdes ved sur pH for at forhindre disulfidudskiftning 13.
Her rapporterer vi en generisk protokol for konjugering af maleimid-funktionaliserede molekyler til et protein, der indeholder en enkelt cysteinrest ved hjælp af reaktionen mellem en Ru (II) -baseret kromofor og redox-proteinet cytochrom c som et eksempel. Denne protokol er lige så anvendelig til de fleste andre proteiner indeholdende en tilgængelig overflade cysteinrest og den tilsvarende maleimid-funktionaliseret mål, det være sig et andet protein, et fluorescerende farvestof, en kromofor eller en syntetisk polymer.
Protocol
Representative Results
Discussion
Oprensning af udgangsmaterialerne før en bioconjugation er af allerstørste betydning. Proteiner opnået fra kommercielle rekombinante kilder indeholder ofte andre isoformer af proteinet af interesse, som kan have forskellige overfladekemi og reaktivitet. For eksempel i den beskrevne bioconjugation, det kommercielt tilgængelige cyt c indeholder en blanding af både iso-1 og iso-2 cyt c 12,14,17. Iso-2 og iso-1 former for cytochrom c er stort set homologe, med de vigtigste forskel …
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.
Materials
| sodium dihydrogen phosphate | Sigma-Aldrich | 71496 | |
| sodium hydroxide | Sigma-Aldrich | 71691 | |
| sodium chloride | Sigma-Aldrich | 73575 | |
| cytochrome c, from saccaromyces cerevisiae | Sigma-Aldrich | C2436 | |
| dithiothreitol | Sigma-Aldrich | 43819 | |
| TSKgel SP-5PW | Sigma-Aldrich | Tosoh SP-5PW, 07161 | 3.3 mL strong cation exchange column |
| Amicon Ultra-15 | Merck-Millipore | UFC900308 | 3.5 kDa spin filter |
| Slide-A-Lyzer mini dialysis units | Thermo Scientific | 66333 | 3.5 kDa dialysis cassetes |
| Ru(II) bisterpyridine maleimide | Lab made | see ref (14) | |
| acetonitrile | Sigma-Aldrich | A3396 | |
| ethylenediaminetetraacetic acid | Sigma-Aldrich | 03609 | |
| tris(2-carboxyethyl)phosphine hydrochloride | Sigma-Aldrich | 93284 | |
| imidazole | Sigma-Aldrich | 56749 | |
| nickel acetate | Sigma-Aldrich | 244066 | |
| AcroSep IMAC Hypercell column | Pall | via VWR: 569-1008 | 1 mL IMAC column |
| 0.2 micron cellulose membrane filter | Whatman | Z697958 | 47 mm filter for buffers |
| 0.2 micron PVDF membrane filter | Merck-Millipore | SLGV013SL | syringe filters for proteins |
| hydrochloric acid | Sigma-Aldrich | 84426 | extremely corrosive! Use caution |
| caffeic acid | Sigma-Aldrich | 60018 | MALDI matrix |
| trifluoroacetic acid | Sigma-Aldrich | 91707 | extremely corrosive! Use caution |
| SimplyBlue SafeStain | Thermo Scientific | LC6060 | Coomassie blue solution |
| NuPAGE Novex 12% Bis-Tris Gel | Thermo Scientific | NP0342BOX | precast protein gels |
| SeeBlue Plus2 Pre-stained Protein Standard | Thermo Scientific | LC5925 | premade protein ladder |
| NuPAGE LDS Sample Buffer (4X) | Thermo Scientific | NP0008 | premade gel sample buffer |
| NuPAGE Sample Reducing Agent (10X) | Thermo Scientific | NP0004 | premade gel reducing agent |
| NuPAGE MES SDS Running Buffer (20X) | Thermo Scientific | NP0002 | premade gel running buffer |
| Voyager DE STR MALDI reflectron TOF MS | Applied Biosystems | ||
| Acta FPLC | GE | Fast Protein Liquid Chromatography | |
| Cary 50 Bio Spectrophotometer | Varian-Agilent | UV-Vis | |
| Milli-Q ultrapure water dispenser | Merck-Millipore | ultrapure water | |
| Low volume UV-Vis Cuvette | Hellma | 105-201-15-40 | 100 microliter cuvette |
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