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 specific labelling to occur. If a protein of interest has multiple cysteine residues, the same protocol applies, albeit with the loss of specificity and product homogeneity. Chemistry targeting surface lysine residues, using N-hydroxysuccinimidyl esters or isothiocyanates, may be a simpler approach if specificity is not required.
<img alt="Figure 1" src="/files/ftp_upload/54157/54157fig1.jpg" /> 
Figure 1. Bioconjugation Reaction Scheme. As an example case, a light harvesting, ruthenium-based antenna molecule will be attached to cytochrome c via Michael addition of a pendant maleimide on the ruthenium-based antenna molecule and an exposed cysteine residue (CYS102) on the protein. The red area of the cyt c surface indicates the heme group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Purification of Cytochrome c
Note: This step is not applicable to all proteins. However, it is important to know that a protein obtained from a commercial supplier can contain other, undesired protein isoforms which may need to be removed by further purification13.
<ol>
	<li>Dissolve 2.4 g of sodium dihydrogen phosphate (Mw = 120 g mol-1) in 1 L of ultrapure water to prepare a buffer solution containing 20 mM NaH2PO4. Adjust the pH with 1 M NaOH to pH 7. 
	Note: The phosphate buffers used in this protocol should be prepared freshly on a daily basis, and filtered through a 0.2 &#181;m cellulose membrane filter prior to use.</li>
	<li>Dissolve 29.22 g of sodium chloride (Mw = 58.44 g mol-1) in 500 ml of the 20 mM NaH2PO4 buffer to make a 20 mM NaH2PO4 and 1 M NaCl buffer. This is the elution buffer for the purification in step 1.6).</li>
	<li>Dissolve 12.0 mg of lyophilized cytochrome c (cyt c) in 6 ml of pH 7 20 mM phosphate buffer.</li>
	<li>Separately dissolve 14.7 mg of dithiothreitol (DTT, Mw = 154.25 g/mol) in 95.3 &#181;l of ultrapure water to prepare a 1 M stock solution. 
	Note: The DTT stock solution should be prepared freshly, as this reagent is susceptible to oxidative deactivation in aqueous solution.</li>
	<li>Pipette 60 &#181;l of the 1 M DTT solution into the protein solution to reduce the cyt c. The color of the solution will change from dark red to light red upon mixing.</li>
	<li>Filter the reduced protein solution through a 0.22 &#181;m low protein binding PVDF syringe filter before injecting onto any chromatographic media.</li>
	<li>Attach a 3.3 ml strong cation exchange column between the injection loop and UV-Vis detector of a Fast Protein Liquid Chromatography (FPLC) instrument.</li>
	<li>Equilibrate the column with 3 column volumes of ultrapure water, followed by 3 column volumes of 20 mM pH 7 phosphate buffer, using a flow rate of 1 ml/min.</li>
	<li>Load 1 ml of reduced crude cyt c into the injection loop, and start a gradient method from 328 - 450 mM NaCl, over 5 column volumes. Monitor the 280 nm and 410 nm channels of the UV-Vis detector, and collect the largest peak.</li>
	<li>Increase the salt concentration to 1 M for 2 column volumes to elute iso-2 cytochrome c, and after the column has been flushed, re-equilibrate the column with 2 column volumes of 20 mM phosphate buffer before injecting the next crude aliquot.</li>
	<li>Repeat steps 1.9-1.10 until all the crude protein has been purified.</li>
	<li>Pool the iso-1 containing fractions and concentrate them using a 3.5 kDa Molecular Weight Cutoff (MWCO) spin filter and centrifuging at 3,000 x g.</li>
	<li>Load the concentrated protein into 3.5 kDa MWCO dialysis cassettes and dialyze against ultrapure water overnight, with 2 changes of water.</li>
	<li>Determine the concentration of the pure, concentrated protein solution by taking 10 &#181;l, diluting it to 100 &#181;l with ultrapure water, and taking an absorbance spectrum. Use a low volume, 100 &#181;l quartz cuvette to obtain protein absorbance spectra. Typically, the undiluted protein concentration is on the order of 50 - 100 &#181;M.
	<ol>
		<li>Use the characteristic 410 nm peak of cyt c to quantify the concentration using the Beer-Lambert law with a molar absorptivity value of 97.6 cm-1 mM-1: 
		A = &#949;&#160;&#215; c&#160;&#215; &#953; 
		where A is the absorbance,&#160;&#949; is the molar absorptivity, c is the concentration in mM, and&#160;&#953; is the path length of the cuvette in cm13.</li>
	</ol>
	</li>
	<li>At this point, divide the protein into 1 ml aliquots and keep frozen at -20 &#176;C until they are needed. Cyt c can be frozen and thawed without loss of structure or function, but repeat cycles will begin to denature the protein. 
	Note: Proteins tolerate freezing to different degrees. For example, green fluorescent proteins5,9 should not be frozen, instead stored in a refrigerator.</li>
</ol>
2. Synthesis of Cytochrome c Bioconjugates
<ol>
	<li>Dissolve 0.9 mg of ruthenium(II) bisterpyridine maleimide (Ru(II) (tpy)2-maleimide, 0.975 &#956;mol, 6 equivalents) in 600 &#956;l of acetonitrile. 
	Note: If the maleimide used is soluble in water, prepare a stock solution in water instead of acetonitrile. It is important for the maleimide to be soluble in the reaction buffer. Dimethylsulfoxide, N,N-dimethylformamide, and acetonitrile are all commonly used adjuvants to assist in the dissolution of low molecular weight12, often hydrophobic maleimide reactants.</li>
	<li>Prepare a buffer solution containing 100 mM phosphate buffer and 100 mM ethylenediaminetetraacetic acid (EDTA, Mw = 292.24 g/mol), which when diluted to 20 mM is at pH 7. Add solid sodium hydroxide until the EDTA is dissolved (several grams typically) and adjust the pH to 7.</li>
	<li>Dissolve 2.87 mg of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP15, Mw = 286.65 g/mol) in 1 ml of ultrapure water to prepare a 10 mM stock solution. This step is important to ensure that the cysteine on the protein is fully reduced prior to maleimide coupling15. 
	Note: The TCEP stock solution should be prepared freshly for each reaction, as this reagent is susceptible to oxidative deactivation in aqueous solution.</li>
	<li>Combine 11.4 ml of ultrapure water with 3 ml of 100 mM phosphate/EDTA stock solution in a 50 ml plastic tube. When diluted further with protein and maleimide stock solutions the buffer concentration will be 20 mM. 
	Note: If the protein being labelled is a transmembrane protein, ensure that a suitable detergent is added to the buffer to solubilize the protein. If a detergent is not used the membrane protein may slowly precipitate, which will negatively impact reaction yields. Use the detergent in all subsequent purification steps.</li>
	<li>Add 0.15 &#181;mol (3 ml of a 50 &#181;M solution, 1 equivalent) of purified cyt c to the phosphate-EDTA buffer.</li>
	<li>Add 7.5 &#181;l of the TCEP stock solution (0.075 &#181;mol, 0.5 equivalents) to the protein solution and leave stirring for 5 min to reduce any protein that may have dimerized due to cysteine oxidation. 
	Note: Do not add TCEP if the protein of interest does not dimerize readily. Check this by running a protein gel under non-reducing conditions.</li>
	<li>Add 600 &#181;l of the acetonitrile solution of Ru(II) (tpy)2-maleimide to the reduced, buffered solution of cyt c, and leave the reaction mixture stirring, in the dark, at RT, for 24 hr.</li>
	<li>Concentrate the reaction mixture using 3.5 kDa MWCO spin filters, centrifuging at 3,000 x g, repeating 2-3 times with fresh 20 mM phosphate buffer until the filtrate runs clear. Remove as much unreacted maleimide from the reaction mixture as possible before the next stage of purification. 
	Note: At this point, the crude bioconjugation mixture can be stored in the refrigerator, in the dark. It is important to remove unreacted maleimide dye by centrifuge dialysis before storage as the maleimide can slowly and nonspecifically react with lysine residues on the surface of the protein.</li>
</ol>
3. Purification of Cytochrome c Bioconjugates
<ol>
	<li>Dissolve 2.4 g of sodium dihydrogen phosphate and 29.22 g of sodium chloride in 1 L of ultrapure water to prepare a buffer solution containing 20 mM NaH2PO4 and 0.5 M NaCl. This is the running buffer for the immobilized metal-affinity chromatography (IMAC) purification.</li>
	<li>Dissolve 17 g of imidazole (Mw = 68.077 g mol-1) in 500 ml of the 20 mM NaH2PO4 and 0.5 M NaCl buffer. This is the elution buffer for the IMAC purification.</li>
	<li>Adjust the pH of both buffers to pH 7 using 1 M NaOH or HCl, and filter them through 0.22 &#181;m regenerated cellulose membranes before use in the FPLC.</li>
	<li>As the IMAC columns purchased are shipped without any metal ions loaded onto the column, prepare the column for a Ni2+-based purification by washing the column with 3 ml of ultrapure water, 3 ml of 100 mM nickel acetate solution, and 6 ml of ultrapure water. If the column is not to be used immediately wash through 3 ml of 20% ethanol and store the column in the refrigerator to prevent bacterial growth.</li>
	<li>Attach a Ni2+-loaded 1 ml IMAC column to the FPLC between the injection loop and UV-Vis detector.</li>
	<li>Equilibrate the column with 3 column volumes of 20 mM phosphate, 0.5 M NaCl buffer using a flow rate of 0.5 ml/min.</li>
	<li>Load 100 &#181;l of crude reaction mixture (filtered through a 0.22 &#181;m syringe filter) on the Ni2+ IMAC column. Wash the column with 3 column volumes of running buffer to elute non-reacted cyt c, followed by an imidazole gradient from 0 to 125 mM over 3 column volumes to elute the ruthenium bisterpyridine-cytochrome c bioconjugate (Ru(II)-cyt c).</li>
	<li>Wash the column with 250 mM imidazole buffer for 5 column volumes, then re-equilibrate the column with 20 mM phosphate, 0.5 M NaCl and repeat step 3.7 until all the crude bioconjugate has been purified.</li>
	<li>Pool the bioconjugate fractions and concentrate them using a 3.5 kDa MWCO spin filter, centrifuging at 3,000 x g.</li>
	<li>Load the concentrated bioconjugate into 3.5 kDa MWCO dialysis cassettes and dialyze against ultrapure water overnight, with 2 changes of water.</li>
	<li>Determine the concentration of the pure, concentrated bioconjugate solution by UV-Vis, using the same molar absorptivity value as iso-1 cytochrome c (97.6 mM-1 cm-1) at 410 nm. Typically, the bioconjugate concentration is on the order of 50 - 100 &#181;M.</li>
	<li>Divide the bioconjugate into 25 &#181;l aliquots and keep frozen at -20 &#176;C until they are needed.</li>
</ol>
4. Characterization of Cytochrome c Bioconjugates
<ol>
	<li>Determination of bioconjugate mass by MALDI-TOF MS
	<ol>
		<li>Dissolve 10 mg of caffeic acid in 1 ml of an acetonitrile/water/trifluoroacetic acid solution (80:20:0.1, v/v/v).</li>
		<li>Dilute 5 &#181;l of concentrated protein solution with 5 &#181;l of caffeic acid solution.</li>
		<li>Spot 0.5 &#181;l of caffeic acid solution on the MALDI target plate and allow the solution to dry.</li>
		<li>Spot 0.5 &#181;l the sample/matrix solution on top of this caffeic acid spot, allow the spot to dry. Spot another 0.5 &#181;l of sample matrix on top of this to &#34;sandwich&#34; the sample between layers of matrix, and allow to dry.</li>
		<li>Acquire mass spectra in linear mode using suitable instrument settings for proteins16.</li>
	</ol>
	</li>
	<li>Investigation of bioconjugates by gel electrophoresis
	<ol>
		<li>Prepare 10 &#181;l of each protein sample to be run by diluting protein samples with premixed lithium dodecyl sulfate (LDS, pH 8.4) buffer (4x) to a final concentration of approximately 20 &#181;g per well. For wells that require reducing conditions, add 1 &#181;l of 500 mM dithiothreitol (DTT) reducing agent (10x).</li>
		<li>Heat samples at 70 &#176;C for 10 min.</li>
		<li>Add 50 ml of premixed 1 M MES, 1 M Tris base, 2% SDS, 20 mM EDTA (pH 7.7) running buffer mixture (20x) from a commercial source to 950 ml of ultrapure water to prepare the gel running buffer.</li>
		<li>Remove the plastic comb from the gel and place the gel in the gel running tank. Fill up the tank with gel running buffer so that the wells are covered with buffer.</li>
		<li>Load samples carefully onto a precast 12% Bis-Tris, 1 mm, 10-well gel using long pipette tips to assist in loading. Load the prestained 10 protein molecular weight marker (3-188 kDa) into a middle well of the gel to aid analysis.</li>
		<li>Run the gel at 200 V constant voltage for 35 min.</li>
		<li>Stain the gel with a commercial Coomassie blue solution for 2 hr, and wash with ultrapure water for 48 hr.</li>
	</ol>
	</li>
	<li>Determination of bioconjugate purity by UV-Vis spectroscopy
	<ol>
		<li>Prepare 120 &#181;l of 5 &#181;M solutions of Ru(II) (tpy)2-maleimide, iso-1 cyt c, and Ru(II)-cyt c.</li>
		<li>Measure the baseline spectrum of the quartz cuvette containing only ultrapure water, from 250 nm to 650 nm.</li>
		<li>Measure the spectrum of each component, ensuring the cuvette is rinsed and dried between each measurement.</li>
		<li>Plot the absorbance of each component as a function of wavelength, and compare the linear sum of the starting materials with the final product to determine if a 1:1 ratio of Ru(II) (tpy)2-maleimide to cyt c has reacted.</li>
	</ol>
	</li>
</ol>

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<li>Li, J., Xu, Q., Cortes, D. M., Perozo, E., Laskey, A., Karlin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactions+of+cysteines+substituted+in+the+amphipathic+N-terminal+tail+of+a+bacterial+potassium+channel+with+hydrophilic+and+hydrophobic+maleimides%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (18), 11605-11610 (2002).</li>
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<li>Borges, C. R., Sherma, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+for+the+Analysis+of+Cysteine+Sulfhydryls+and+Oxidative+Protein+Folding%5BTitle%5D)+AND+%22Antioxid.+Redox+Signal%22%5BJournal%5D)">Techniques for the Analysis of Cysteine Sulfhydryls and Oxidative Protein Folding.</a> Antioxid. Redox Signal. (3), 1-21 (2014).</li>
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The authors have nothing to disclose.

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 the presence of a free cysteine residue near the C-terminus of iso-1 cytochrome c. In the example here, purification is achieved with aqueous strong cation-exchange FPLC, however, other forms of FPLC such as anion exchange, affinity, hydrophobic or size-exclusion chromatography may be more applicable. The protein is also reduced in this step to ensure that the cysteine residue on the protein of interest (here cytochrome c) is fully reduced prior to bioconjugation with the maleimide-linked target. In addition, it is useful to ascertain if the maleimide-appended small molecule to be used with a cysteine containing protein is pure and that the maleimide has not undergone a reaction in storage, as maleimides are light and temperature sensitive. This can be quickly and easily checked by 1H NMR, as the vinylic protons of an intact maleimide will appear as a singlet at 6-6.5 ppm, whereas the protons of an open maleimide will shift upfield; and also mass spectrometry, with an open maleimide ring corresponding to an 18 Da mass increase.
Buffer choice, pH, and the inclusion of adjuvants such as detergents, organic solvents, and reducing agents will have a profound effect on bioconjugation yields5,15. In the case of a Michael addition of a maleimide and a thiol, the pH must be above 6 yet below 8 for a fast, specific reaction to occur. Below pH 6, specificity is high as the maleimide will not be able to react with any amines, but the thiol is protonated and thus is a poor nucleophile for Michael addition leading to a slow reaction. Above pH 8, surface lysine residues can become deprotonated and react with available maleimides, leading to a non-specific covalent attachment of the maleimide component to the protein. Phosphate buffer is used in this bioconjugation because it is a strong buffer in this pH range and does not interact with any of the reagents. Tris(hydroxymethyl) aminomethane (Tris) buffer, for example, would be a poor choice as the amine group in this buffer could potentially react with the maleimide, which would lead to poor yields. Solubility of all reagents is also key to high yields. The addition of small amounts (&#60;10% v/v) of organic solvent can assist in the dissolution of non-polar components, while correct pH and salt concentration are essential for keeping proteins in solution and folded12. For large membrane proteins with significant hydrophobic surface residues, a surfactant will be necessary to prevent the protein from precipitating18.
If the protein in the bioconjugation is prone to oxidation and dimerization, the addition of a reducing agent such as TCEP has been shown to improve bioconjugation yields15. The in situ reduction of the protein to its monomeric, free-thiol form allows a greater proportion of active thiol sites to be accessed by the maleimide. However, order of addition and stoichiometry is the key to a successful increase in yield as TCEP will also react with maleimide. By adding the maleimide after TCEP, the TCEP will be consumed by reducing the protein first. Half an equivalent of TCEP to protein is used for the same reason, so that excess TCEP is not available to react unfavorably with the free maleimide. Structural disulfide bridges on the interior of the protein are not likely to be affected by the addition of TCEP or other reducing agents such as DTT, provided that the protein is not held in denaturing conditions such as high temperature or a high concentration of urea. For example, the addition of a large excess of DTT to cyt c prior to FPLC purification does not have a significant effect on the amount of protein recovered after purification. If reduction of structural cysteine bridges by TCEP is suspected, this can be confirmed by running a protein gel under native, non-denaturing conditions.
The Ru(II) bisterpyridine-labelled cytochrome c synthesized in this method is purified via Ni2+ Immobilized Metal Affinity Chromatography14. Other purification methods exist, such as size exclusion chromatography, anion/cation exchange chromatography, and specific affinity chromatography such as an antibody stationary phase. In general, the method remains the same as described above, with concentration and dialysis steps following chromatographic purification to return the bioconjugate product to a suitable buffer for storage.
The primary method of determining if a bioconjugation is successful is via MALDI-TOF MS. It is the most accurate way to ascertain the small difference in mass between native protein and protein covalently labelled with a small molecule, a difference often less than 1,000 Da. The most difficult part of running a MALDI-TOF MS experiment is matrix choice and spotting technique; however caffeic acid has been found to be a reliable, general purpose matrix for the analysis of the proteins and bioconjugates used in this work. As a potential alternative, sinapinic acid is another commonly used MALDI matrix for the analysis of proteins. Analysis of MALDI-TOF MS data is relatively straightforward. Figure 2 shows the typical MALDI-TOF MS spectrum of both pure iso-1 cyt c (Mw = 12,706 Da) and pure Ru(II)-cyt c (Mw = 13,559 Da), both of which closely correspond to the calculated molecular mass. The purity can also be observed, noting the lack of an iso-2 cytochrome c peak (Mw = 12,532 Da) in the unreacted spectrum and the lack of an iso-1 cytochrome c peak in the bioconjugate spectrum. The bioconjugate mass can also be estimated using gel electrophoresis. Figure 3, a 12% Bis-Tris protein gel, provides two pieces of evidence for bioconjugation. First, the absence of a dimer band under non-reducing conditions for Ru(II)-cyt c indicates that there is no longer a free cysteine, and second, the slight shift of the bioconjugate band upwards translates to an increase in molecular weight. Protein gels are invaluable and can definitively provide proof of successful bioconjugation not only for small molecule attachments, but also for the synthesis of protein dimers5 or protein-polymer bioconjugates9.
For proteins containing chromophores such as green fluorescent protein or heme-containing cytochromes, UV-Vis spectroscopy is used to determine the composition and yield of the bioconjugate. The 1:1 linear addition of the starting materials spectra allows the construction of a hypothetical UV-Vis spectrum of the bioconjugate, as shown in Figure 4. By comparing the actual bioconjugate spectrum to this 1:1 addition spectrum, the composition can be inferred. For example, if two or more Ru(II) (tpy)2-maleimides had nonspecifically attached to the protein, the 480 nm band of the bioconjugate would be higher than the predicted spectrum. In addition, the concentration of the bioconjugate can be approximated by using the 410 nm molar absorptivity value of 97.6 cm-1 mM-1. This is assumed to be the same for the bioconjugate because the Ru(II) (tpy)2-maleimide has minimal absorbance in this region.
It should be noted that bioconjugation via cysteine-maleimide chemistry has several limitations. Firstly, the protein of interest must have a single, accessible cysteine residue, or else protein engineering must be performed to introduce one. Secondly, the cysteine-maleimide bond is susceptible to exchange with other free thiols, such as albumin and glutathione in the context of plasma applications19. Such exchange is highly dependent on the solvent accessibility and local charge on the surface of the protein. Maleimide-thiol linkages may still be suitable for plasma applications, but long term stability should be checked using mass spectrometry and gel electrophoresis.
Despite this, the highly specific, high yielding attachment of novel molecules such as fluorescent dyes, redox centers, polymers, and other proteins to proteins via cysteine-maleimide chemistry is a powerful technique that allows a range of interesting macromolecular constructs to be accessed. Having large quantities of chemically well-defined protein bioconjugates is key to future studies involving protein binding, self-assembly, enzyme kinetics and protein localization. As demonstrated above, starting material purification, choice of reaction medium, and the addition of supplementary reagents such as reducing agents or surfactants all have a significant impact on bioconjugation yields. Purification is most easily achieved by protein-compatible chromatography, and characterization is accomplished using a combination of MALDI-TOF MS, gel electrophoresis, and UV-Vis spectroscopy.

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><tbody><tr><td>sodium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>71496</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>71691</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma-Aldrich</td><td>73575</td><td></td></tr><tr><td>cytochrome &lt;em&gt;c&lt;/em&gt;, from saccaromyces cerevisiae</td><td>Sigma-Aldrich</td><td>C2436</td><td></td></tr><tr><td>dithiothreitol</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>TSKgel SP-5PW</td><td>Sigma-Aldrich</td><td>Tosoh SP-5PW, 07161</td><td>3.3 ml strong cation exchange column</td></tr><tr><td>Amicon Ultra-15&amp;nbsp;</td><td>Merck-Millipore</td><td>UFC900308</td><td>3.5 kDa spin filter</td></tr><tr><td>Slide-A-Lyzer mini dialysis units</td><td>Thermo Scientific</td><td>66333</td><td>3.5 kDa dialysis cassetes</td></tr><tr><td>Ru(II) bisterpyridine maleimide</td><td>Lab made</td><td></td><td>see ref (14)</td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>A3396</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>03609</td><td></td></tr><tr><td>tris(2-carboxyethyl)phosphine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>93284</td><td></td></tr><tr><td>imidazole</td><td>Sigma-Aldrich</td><td>56749</td><td></td></tr><tr><td>nickel acetate</td><td>Sigma-Aldrich</td><td>244066</td><td></td></tr><tr><td>AcroSep IMAC Hypercell column</td><td>Pall</td><td>via VWR: 569-1008</td><td>1 ml IMAC column</td></tr><tr><td>0.2 micron cellulose membrane filter</td><td>Whatman</td><td>Z697958</td><td>47 mm filter for buffers</td></tr><tr><td>0.2 micron PVDF membrane filter</td><td>Merck-Millipore</td><td>SLGV013SL</td><td>syringe filters for proteins</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>84426</td><td>Extremely corrosive! Use caution.</td></tr><tr><td>caffeic acid</td><td>Sigma-Aldrich</td><td>60018</td><td>MALDI matrix</td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td>extremely corrosive! Use caution</td></tr><tr><td>SimplyBlue SafeStain</td><td>Thermo Scientific</td><td>LC6060</td><td>Coomassie blue solution</td></tr><tr><td>NuPAGE Novex 12% Bis-Tris Gel</td><td>Thermo Scientific</td><td>NP0342BOX</td><td>precast protein gels</td></tr><tr><td>SeeBlue Plus2 Pre-stained Protein Standard</td><td>Thermo Scientific</td><td>LC5925</td><td>premade protein ladder</td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Thermo Scientific</td><td>NP0008</td><td>premade gel sample buffer</td></tr><tr><td>NuPAGE Sample Reducing Agent (10x)</td><td>Thermo Scientific</td><td>NP0004</td><td>premade gel reducing agent</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Thermo Scientific</td><td>NP0002</td><td>premade gel running buffer</td></tr><tr><td>Voyager DE STR MALDI reflectron TOF MS</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Acta FPLC</td><td>GE</td><td></td><td>Fast Protein Liquid Chromatography</td></tr><tr><td>Cary 50 Bio Spectrophotometer</td><td>Varian-Agilent</td><td></td><td>UV-Vis</td></tr><tr><td>Milli-Q ultrapure water dispenser</td><td>Merck-Millipore</td><td></td><td>ultrapure water</td></tr><tr><td>Low volume UV-Vis Cuvette</td><td>Hellma</td><td>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-maleimide to cyt c and subsequent purification of the bioconjugate. The UV-Vis spectrum of the bioconjugate allows the yield to be calculated with the absorbance at 410 nm, and by comparing the spectrum to a predicted 1:1 addition spectrum of the starting materials the composition of the bioconjugate can be inferred. In the example above, the yield varies somewhat from batch-to-batch but it generally between 15-27% after FPLC purification15.
In addition, the appearance of a new peak in the chromatogram during purification confirms the synthesis of a new species. This is exemplified in Figure 5, where analysis of the different UV-Vis traces can indicate whether or not a species contains the Ru(II) (tpy)2-maleimide component.

<img alt="Figure 2" src="/files/ftp_upload/54157/54157fig2.jpg" /> 
Figure 2. MALDI-TOF Mass Spectra of Proteins. Mass spectra of pure iso-1 cytochrome c (black) and Ru(II)-cyt c (red). Peaks can be observed that correspond to the calculated masses of iso-1 cyt c (12,706 Da) and Ru(II)-cyt c (13,559 Da) with a caffeic acid adduct visible at +179 Da. This adduct is seen in higher amounts in the unreacted cyt c spectra due to a reaction between the &#945;, &#946;-unsaturated carbonyl of caffeic acid and the free thiol of the protein under high-energy MALDI conditions. Matrix adducts are commonly seen in MALDI-MS and can be both covalent and non-covalent in nature. Spectra are baseline corrected, noise filtered, and normalized for comparison. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54157/54157fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54157/54157fig3.jpg" /> 
Figure 3. SDS-PAGE of Proteins. 12% Bis-Tris gel of reduced and non-reduced cyt c and Ru(II)-cyt c. Lane 3 contains a pre-stained protein standard, with polypeptide mass annotated to the right of each band (kDa). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54157/54157fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54157/54157fig4.jpg" /> 
Figure 4. UV-Vis Spectra of Proteins: The absorbance spectrum of Ru(II)-cyt c (green) corresponds closely to the linear addition (dashed blue) of pure, unreacted cyt c (red) and unreacted Ru(II) (tpy)2-maleimide (black). This demonstrates that the bioconjugate consists of a 1:1 attachment of the maleimide to the protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54157/54157fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54157/54157fig5.jpg" /> 
Figure 5. Chromatogram of Ni-IMAC purification of bioconjugates: IMAC trace of Ru(II)-cyt c purification. UV-Vis traces: 410 nm corresponds to the Soret band of cyt c, 280 nm corresponds to both cyt c and Ru(II) (tpy)2-maleimide, and 475 nm corresponds to the metal-to-ligand charge transfer band of the ruthenium(II) complex. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54157/54157fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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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38.5K Views.  The University of New South Wales.  Dit protocol geeft de belangrijkste stappen voor de bioconjugatie van een cysteïne bevattend eiwit een maleïmide, zoals zuivering reagens, reactieomstandigheden, bioconjugaat zuivering en karakterisering bioconjugaat. 

Video: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Immunology and Infection

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

Biochemistry

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Bioengineering

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

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