Published: July 20th, 2016
This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.
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.
Bioconjugation involves covalently linking one biomolecule with another or with a synthetic molecule such as a dye, drug or a polymer. Protein bioconjugation methods are now extensively used in many chemistry, biology and nanotechnology research groups with applications ranging from fluorescent dye labelling1,2, making of protein (antibody)-prodrugs3 (antibody drug conjugates — ADCs) synthesis of protein dimers4,5, through to self-assembling protein-polymer hybrids6,7 used in nanomedicine8 and systems chemistry9.
Specificity of the chemistry used for bioconjugation, whi....
Note: The following protocol is designed for the synthesis of a protein-dye bioconjugate as shown in Figure 1. It is a general protocol for the reaction of a maleimide with free surface cysteine containing proteins, with notes inserted where applicable to assist with membrane protein bioconjugates, protein-polymer bioconjugates, and synthetic protein dimer (protein-protein) bioconjugates. In this particular case, the protein iso-1 cytochrome c has one surface cysteine residue available to react which allows a highly.......
The synthesis of bioconjugates is confirmed by three primary methods: Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), polyacrylamide gel electrophoresis, and Ultraviolet-Visible (UV-Vis) spectroscopy, as shown in Figures 2, 3 and 4. A mass increase corresponding to the mass of the appended small molecule, and the lack of an unreacted protein demonstrates the successful covalent linkage of Ru(II) (tpy)2
Purification of the starting materials before a bioconjugation is of utmost importance. Proteins obtained from commercial recombinant sources often contain other isoforms of the protein of interest, which can have different surface chemistry and reactivity. For example, in the described bioconjugation, the commercially available cyt c contains a mixture of both iso-1 and iso-2 cyt c12,14,17. Iso-2 and iso-1 forms of cytochrome c are largely homologous, with the main difference being .......
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.....
|sodium dihydrogen phosphate
|cytochrome c, from saccaromyces cerevisiae
|Tosoh SP-5PW, 07161
|3.3 mL strong cation exchange column
|3.5 kDa spin filter
|Slide-A-Lyzer mini dialysis units
|3.5 kDa dialysis cassetes
|Ru(II) bisterpyridine maleimide
|see ref (14)
|AcroSep IMAC Hypercell column
|via VWR: 569-1008
|1 mL IMAC column
|0.2 micron cellulose membrane filter
|47 mm filter for buffers
|0.2 micron PVDF membrane filter
|syringe filters for proteins
|extremely corrosive! Use caution
|extremely corrosive! Use caution
|Coomassie blue solution
|NuPAGE Novex 12% Bis-Tris Gel
|precast protein gels
|SeeBlue Plus2 Pre-stained Protein Standard
|premade protein ladder
|NuPAGE LDS Sample Buffer (4X)
|premade gel sample buffer
|NuPAGE Sample Reducing Agent (10X)
|premade gel reducing agent
|NuPAGE MES SDS Running Buffer (20X)
|premade gel running buffer
|Voyager DE STR MALDI reflectron TOF MS
|Fast Protein Liquid Chromatography
|Cary 50 Bio Spectrophotometer
|Milli-Q ultrapure water dispenser
|Low volume UV-Vis Cuvette
|100 microliter cuvette
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