Name: synthesis of protein bioconjugates via cysteine-maleimide chemistry
Uploaded: 2016-07-20T15:00:00
Description: Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

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.

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...

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A., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+room+temperature+photo-induced+electron+transfer+in+biologically+active+bis(terpyridine)ruthenium(II)-cytochrome+c+bioconjugates+and+the+effect+of+solvents+on+the+bioconjugation+of+cytochrome+c.">Synthesis and room temperature photo-induced electron transfer in biologically active bis(terpyridine)ruthenium(II)-cytochrome c bioconjugates and the effect of solvents on the bioconjugation of cytochrome c.</a> Org. Biomol. Chem. 8, 151-162 (2010).</li><li>Borges, C. R., Sherma, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+the+Analysis+of+Cysteine+Sulfhydryls+and+Oxidative+Protein+Folding.">Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding.</a> Antioxid. Redox Signal. 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Biochem. 31, 139-143 (1972).</li><li>M&#252;ller, M., Azzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+beef+heart+cytochrome+oxidase+subunit+III+with+eosin-5-maleimide.">Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.</a> FEBS Lett. 184 (1), 110-114 (1985).</li><li>Shen, B. Q., 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=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat. Biotechnol. 30 (2), 184-189 (2012).</li></ol>

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 ...

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 &#8212; 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, while not always critical, is of utmost importance for most functional protein bioconjugates, so as to not interfere with the active site of the target protein. The ideal bioconjugation reaction needs to fulfill several criteria, including: i) targeting rare or unique sites on the protein of interest, ii) be selective towards this target, iii) proceed under non-denaturing conditions to avoid protein unfolding and iv) be high-yielding as the target protein is usually only available at sub-millimolar concentration. The maleimide - cysteine Michael addition comes close to fulfilling all these criteria, and has for that reason long claimed a special status in the field of bioconjugate chemistry10. This is because i) many proteins containing only one cysteine residue on their surface can be genetically engineered there, ii) at the correct pH the reaction is highly selective towards cysteine, iii) it proceeds smoothly in aqueous buffers and iv) it is very fast with the second order rate constant of maleimides to cysteine-containing proteins reported to exceed 5,000 M-1 sec-1 in some cases11. Provided the protein of interest can tolerate a small (&#8776; 5-10%) amount of organic co-solvent12, almost any maleimide-functionalized dye, polymer, surface or another protein can be linked to proteins. In addition, maleimides are more specific for cysteines on proteins than iodoacetamides, which are more prone to reacting with other nucleophiles at elevated pH; and more stable than disulfide-based conjugations which need to be kept at acidic pH to prevent disulfide exchange13.
Here we report a generic protocol for the conjugation of maleimide-functionalized molecules to a protein containing a single cysteine residue using the reaction between a Ru(II)-based chromophore and the redox protein cytochrome c as an example. This protocol is equally applicable to most other proteins containing an accessible surface cysteine residue and the corresponding maleimide-functionalized target, be it another protein, a fluorescent dye, a chromophore or a synthetic polymer.

<table border="0" cellpadding="0" cellspacing="0" width="913">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium dihydrogen phosphate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71496</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium hydroxide</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">71691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium chloride</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">73575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">cytochrome c, from saccaromyces cerevisiae</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">C2436</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">dithiothreitol</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">TSKgel SP-5PW</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">Tosoh SP-5PW, 07161</td>
			<td>3.3 mL strong cation exchange column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15&#160;</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">UFC900308</td>
			<td>3.5 kDa spin filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide-A-Lyzer mini dialysis units</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">66333</td>
			<td>3.5 kDa dialysis cassetes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ru(II) bisterpyridine maleimide</td>
			<td style="width:196px;">Lab made</td>
			<td style="width:151px;"></td>
			<td>see ref (14)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">acetonitrile</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">A3396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">ethylenediaminetetraacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">03609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">93284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">imidazole</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">nickel acetate</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">244066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">AcroSep IMAC Hypercell column</td>
			<td style="width:196px;">Pall</td>
			<td style="width:151px;">via VWR: 569-1008</td>
			<td>1 mL IMAC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron cellulose membrane filter</td>
			<td style="width:196px;">Whatman</td>
			<td style="width:151px;">Z697958</td>
			<td>47 mm filter for buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.2 micron PVDF membrane filter</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;">SLGV013SL</td>
			<td>syringe filters for proteins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hydrochloric acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">84426</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">caffeic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">60018</td>
			<td>MALDI matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">trifluoroacetic acid</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:151px;">91707</td>
			<td>extremely corrosive! Use caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC6060</td>
			<td>Coomassie blue solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Novex 12% Bis-Tris Gel</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0342BOX</td>
			<td>precast protein gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SeeBlue Plus2 Pre-stained Protein Standard</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">LC5925</td>
			<td>premade protein ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE LDS Sample Buffer (4X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0008</td>
			<td>premade gel sample buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE Sample Reducing Agent (10X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0004</td>
			<td>premade gel reducing agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">NuPAGE MES SDS Running Buffer (20X)</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td style="width:151px;">NP0002</td>
			<td>premade gel running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Voyager DE STR MALDI reflectron TOF MS</td>
			<td style="width:196px;">Applied Biosystems</td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Acta FPLC</td>
			<td style="width:196px;">GE</td>
			<td style="width:151px;"></td>
			<td>Fast Protein Liquid Chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Cary 50 Bio Spectrophotometer</td>
			<td style="width:196px;">Varian-Agilent</td>
			<td style="width:151px;"></td>
			<td>UV-Vis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Milli-Q ultrapure water dispenser</td>
			<td style="width:196px;">Merck-Millipore</td>
			<td style="width:151px;"></td>
			<td>ultrapure water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Low volume UV-Vis Cuvette</td>
			<td style="width:196px;">Hellma</td>
			<td style="width:151px;">105-201-15-40</td>
			<td>100 microliter cuvette</td>
		</tr>
	</tbody>
</table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

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.

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</su...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry (Video) | JoVE

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.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

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A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction (Video) | JoVE

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides (Video) | JoVE

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile (Video) | JoVE

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers (Video) | JoVE

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...