Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Summary

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.

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 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 labelling^1,2, making of protein (antibody)-prodrugs³ (antibody drug conjugates — ADCs) synthesis of protein dimers^4,5, through to self-assembling protein-polymer hybrids^6,7 used in nanomedicine⁸ and systems chemistry⁹.

Specificity of the chemistry used for bioconjugation, whi....

Protocol

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 specific labelling to occur. If a protein of interest has multiple cysteine residues, the same protocol applies, albeit with the loss of specificity ....

Results

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

Discussion

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 c^12,14,17. Iso-2 and iso-1 forms of cytochrome c are largely homologous, with the main difference being .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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