1. Plasmid Construction
<ol>
	<li>Construct an expression plasmid (pBAD-HerFab-L177TAG) that would express the target antibody gene (pBAD-HerFab-WT) with a His6-tag, and replace the codon for Leucine-177 with the amber codon (TAG) 27, using conventional site-directed mutagenesis technique. 
	See Table of Materials.</li>
	<li>Construct another expression plasmid (pEVOL-AFRS) containing the genes for the evolved tRNATyr and aminoacyl-tRNA synthetase (aaRS) pair. Use the specially designed plasmid vector, pEVOL, for efficient incorporation of UAAs. The detailed plasmid information and cloning protocols are described in the previous report.28</li>
	<li>Obtain high quality plasmid DNA by using commercially available plasmid preparation kits. The 260/280 ratio of ~1.8 is optimal for the purified DNA. If required, perform 1% agarose gel electrophoresis to check the purity of the DNA.</li>
</ol>
2. Culture Preparation
<ol>
	<li>Electroporation
	<ol>
		<li>Add each 1 &#181;L DNA of pEVOL-AFRS28 and pBAD-HerFab-L177TAG to 20 &#181;L Escherichia coli DH10&#946; strain. Mix gently, using a pipette. The plasmids can be transformed together or in independent reactions.</li>
		<li>For 0.1 cm cuvettes, set the electroporation settings to 25 &#181;F and 2.5 kV.</li>
		<li>Insert the cuvette into the slide of the shocking chamber. Push the slide into the chamber until the cuvette makes firm contact with the chamber electrodes.</li>
		<li>Pulse once by pressing the pulse button until the electroporation machine beeps.</li>
		<li>Add 1 mL super optimal broth with catabolite repression (SOC) medium containing 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose to the cuvette quickly and transfer the mixture to a test tube. Incubate the cells for 1 h at 37 &#176;C with shaking.</li>
		<li>Spread transformed E. coli cells on Lysogeny broth (LB) agar plates containing appropriate antibiotics (35 &#181;g/mL chloramphenicol and 100 &#181;g/mL ampicillin for selecting pEVOL-AFRS and pBAD-HerFab-L177TAG, respectively). Incubate the plates at 37 &#176;C for 12 h.</li>
	</ol>
	</li>
	<li>Culture preparation
	<ol>
		<li>Inoculate a single transformed colony in 5 mL LB medium containing antibiotics. Incubate for 12 h at 37 &#176;C with shaking.</li>
	</ol>
	</li>
</ol>
3. Expression and Purification of HerFab-L177AF
<ol>
	<li>Expression of HerFab-L177AF
	<ol>
		<li>Transfer the primary culture (5 mL) into 200 mL LB medium containing ampicillin (100 &#181;g/mL) and AF (1 mM), followed by incubation at 37 &#176;C with shaking. 
		NOTE: 100 mM AF stock solution (10 mL) was prepared by dissolving 206 mg AF in 100 mM hydrochloric acid (10 mL, final volume).</li>
		<li>Add 2 mL of 1.6 M arabinose (16 mM final concentration) when the culture reaches the log-phase (OD550 = 0.8, OD = Optical density). Incubate for 12 h at 30 &#176;C with shaking.</li>
		<li>Harvest cells by centrifugation at 11,000 x g for 5 min. Discard supernatant and freeze the pellet at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Resuspend the cell pellets in 20 mL periplasmic lysis buffer containing 30 mM Tris (pH 8.0), 1 mM EDTA, 20% sucrose, and 0.2 mg/mL lysozyme, and incubate the mixture for 1 h at 37 &#176;C.</li>
		<li>Centrifuge the cell lysate at 18,000 x g at 4 &#176;C for 15 min. Transfer the supernatant to a fresh tube and discard the pellet.</li>
	</ol>
	</li>
	<li>Ni-NTA affinity chromatography
	<ol>
		<li>Add Ni-NTA resin suspension to each centrifuge tube (400 &#181;L resin for a 200 mL culture), and mix gently at 4 &#176;C for 1 h.</li>
		<li>Pour the suspension into a polypropylene column and wash the resin three times with 5 mL wash buffer containing 50 mM NaH2PO4 (pH 8.0), 20 mM imidazole, and 300 mM NaCl.</li>
		<li>Elute the target antibody with 300 &#181;L elution buffer containing 50 mM NaH2PO4 (pH 8.0), 250 mM imidazole, and 300 mM NaCl.</li>
		<li>Determine concentration of the mutant protein by Bradford protein assay29 or by measuring the absorbance at 280 nm. Calculate the extinction coefficient (75,866 M-1cm-1) for HerFab-L177AF at 280 nm by protein extinction coefficient calculator using the extinction coefficient (2,471 M-1cm-1) for AF. 
		NOTE: The amino acid sequence of HerFab can be found in the NCBI website (GI:783282791 and GI:783282792).</li>
	</ol>
	</li>
</ol>
4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-promoted Azide-alkyne Cycloaddition (SPAAC)
<ol>
	<li>Add 20 &#181;L of Cy5.5-Azadibenzocyclooctyne(Cy5.5-ADIBO) in H2O (200 &#181;M final concentration) to a solution of 10 &#181;L of HerFab-L177AF (10 &#181;M final concentration) in phosphate buffer containing 10 mM Na2HPO4 (pH 7.0) and 100 mM NaCl. 
	NOTE: As alternatives, difluorinated cyclooctyne (DIFO) and bicyclo[6.1.0]nonyne (BCN) derivatives are also available for the same application.</li>
	<li>Allow the strain-promoted cycloaddition reaction to proceed for 6 h at 37 &#176;C. If a light-sensitive probe (e.g., Cy5.5) is used, cover the reaction vessel with aluminum foil.</li>
	<li>Purify the labeled HerFab according to step 5.</li>
</ol>
5. Purification of Labeled HerFab
<ol>
	<li>Add 500 &#181;L of sample to a centrifugal filter spin column.</li>
	<li>Centrifuge the spin column at 14,000 x g at 4 &#176;C for 15 min.</li>
	<li>Discard flow-through and transfer purified sample from the spin column to a 1.5 mL microcentrifuge tube.</li>
	<li>As an alternative to step 5.1- 5.3, perform purification by dialysis against the same buffer.</li>
	<li>Store the purified labeled HerFab at 4 &#176;C.</li>
</ol>
6. SDS-PAGE Analysis of Labeled HerFab
<ol>
	<li>Add 5 &#181;L LDS protein sample buffer containing 106 mM Tris&#183;HCl, 141 mM Tris base (pH 8.5), 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, and 0.175 mM phenol red to 13 &#181;L of purified labeled HerFab (7.8 &#181;M or 0.38 mg/mL) in the presence (+) or absence (&#8722;) of 2 &#181;L dithiothreitol (DTT, 100 mM final concentration). Incubate the mixture at 95 &#176;C for 10 min.</li>
	<li>Attach the 4-12% Bis-Tris SDS-PAGE gel cassette to the electrophoresis cell and add running buffer. Load the conjugated protein samples and the pre-stained molecular weight marker. Perform gel electrophoresis for 35 min at 200 V. 
	Note: Keep the electrophoresis cell in the dark during the entire period to minimize photo-bleaching of the fluorophore.</li>
	<li>After electrophoresis, transfer the gel to a fluorescent gel scanner, and scan for fluorescence emission at the appropriate wavelength. For Cy5.5, use the Cy5 mode in the scanner software.</li>
	<li>Stain the gel with a commercial protein stain.</li>
</ol>

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The authors have nothing to disclose.

The genetic incorporation of unnatural amino acids into proteins has several advantages over other methods used for protein modification. 1-3 One of the important advantages is its general applicability to any kind of protein. In principle, there is no limitation in selecting a target protein and a target site of the protein. However, replacement of a structurally or functionally important residue with a UAA may result in altering the structure and function of the target protein. Generally, residues that are exposed to solvent and do not interact with other residues are chosen for incorporation of UAAs. Therefore, structural information from a high-resolution crystal structure is used in order to choose optimal sites for UAA incorporation. 30 Because the incorporation of UAAs is technically easy and requires a simple mutagenesis, multiple sites can be readily screened to select an optimal residue for a desired function of a target protein.
In this protocol, AF is genetically incorporated into an antibody fragment, and the mutant fragment is site-specifically labeled with a fluorophore, using SPAAC. The conjugation yield of this method is quantitative without any undesired reaction, which is an important improvement over the previous report. 25 This was achieved by careful structural analysis for the selection of an optimal site and an increase in the reaction time. Therefore, the choice of the site for UAA incorporation is critical for fast and quantitative conjugation. In addition, the use of efficient UAA incorporation systems (e.g., pEvol-AFRS) is also critical for protein yield and incorporation efficiency. 28
Other UAAs incorporated into proteins by genetic incorporation method for protein conjugation can also be used for the antibody conjugation. 4-11 In terms of reaction rate, copper-catalyzed cycloaddition reaction, as well as inverse electron-demand Diels-Alder reaction using tetrazines8 and strained alkenes or alkynes, 7 will be better than the SPAAC used in this study. However, the copper-catalyzed cycloaddition reaction requires copper(I) ion and ligands, 31 and the amino acids for the Diels-Alder reaction are often synthetically challenging, and not stable enough for quantitative conjugation. Ketone-hydroxyamine condensation4 can also be used for the same purpose. However, it requires moderately acidic conditions, and its reaction rate is slower than that of the SPAAC. Considering the factors such as commercial and synthetic accessibility of unnatural amino acids, the reaction rate, bio-orthogonality, biocompatibility of reaction conditions, and incorporation efficiency of unnatural amino acids, the method of using SPAAC and genetically incorporated AF would be useful for developing modified therapeutic proteins such as ADCs, as well as for general protein labeling.

Since the genetic incorporation of p-methoxyphenylalanine in Escherichia coli was reported,1 more than 100 unnatural amino acids (UAAs) have been successfully incorporated into various proteins.1-3 Among these UAAs, the amino acids containing bioorthogonal functional groups have been extensively studied and represent the largest proportion. The bioorthogonal functional groups used in the UAAs include ketone,4 azide,5 alkyne,6 cyclooctyne,7 tetrazine,8 &#945;,&#946;-unsaturated amide,9 norbonene,10 transcyclooctene,11 and bicyclo[6.1.0]-nonyne.11 Although each functional group has its advantages and disadvantages, the azide-containing amino acids have been most extensively used for protein conjugation. p-Azidophenylalanine (AF), one of the azido-containing amino acids, is readily available, and its incorporation efficiency is excellent. Mutant proteins containing this amino acid can be reacted with alkynes by copper-catalyzed cycloaddition or with cyclooctynes by SPAAC.12-20
Recently, biopharmaceuticals have been attracting great attention in the pharmaceutical industry. The antibody-drug conjugate (ADC) is a class of therapeutic antibodies that are advantageous due to their ability for targeted therapy for the treatment of human cancers21 and other diseases. More than 50 ADCs are currently in clinical trials, and the number is rapidly increasing. In development of ADCs, many factors need to be considered to maximize the efficacy and minimize the side effects. Among these factors, an efficient and site-specific conjugation reaction to form a covalent bond between an antibody and a drug is critical. The desired efficiency and specificity in the conjugation reaction can be achieved by conjugation with a bioorthogonal functional group in an unnatural amino acid that is specifically incorporated into an antibody.22-26 Here, we report a protocol to site-specifically incorporate AF into an antibody fragment and conjugate the mutant antibody fragment with a biochemical probe.

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. Plasmid Construction&lt;/strong&gt;</td></tr><tr><td>plasmid pBAD_HerFab_L177TAG</td><td></td><td></td><td>Optionally contain the amber stop codon (TAG) at a desired position. Ko, W. &lt;em&gt;et al.&lt;/em&gt; Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. &lt;em&gt;BKCS.&lt;/em&gt; &lt;strong&gt;36 (9)&lt;/strong&gt;, 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td></tr><tr><td>plasmid pEvol-AFRS</td><td></td><td></td><td>Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in &lt;em&gt;E. coli.&lt;/em&gt; &lt;em&gt;J. Mol. Biol.&lt;/em&gt; &lt;strong&gt;395 (2)&lt;/strong&gt;, 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)</td></tr><tr><td>DH10B</td><td>Invitrogen</td><td>C6400-03</td><td>Expression Host</td></tr><tr><td>Plasmid Mini-prep kit</td><td>Nucleogen</td><td>5112</td><td>200/pack</td></tr><tr><td>Agarose</td><td>Intron biotechnology</td><td>32034</td><td>500&amp;nbsp;g</td></tr><tr><td>Ethidium bromide</td><td>Alfa Aesar</td><td>L07482</td><td>1 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500&amp;nbsp;g</td></tr><tr><td colspan="4">&lt;strong&gt;2. Culture Preparation&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;2.1 Electroporation&lt;/strong&gt;</td></tr><tr><td>Micro pulser</td><td>BIO-RAD</td><td>165-2100</td><td></td></tr><tr><td>Micro pulser cuvette</td><td>BIO-RAD</td><td>165-2089</td><td>0.1&amp;nbsp;cm electrode gap, pkg. of 50</td></tr><tr><td>Ampicillin Sodium</td><td>Wako</td><td>018-10372</td><td>25 g</td></tr><tr><td>Chloramphenicol</td><td>Alfa Aesar</td><td>B20841</td><td>25 g</td></tr><tr><td>Agar</td><td>SAMCHUN</td><td>214230</td><td>500 g</td></tr><tr><td>SOC medium</td><td>Sigma</td><td>S1797</td><td>100 mL</td></tr><tr><td colspan="4">&lt;strong&gt;3. Expression and Purification of HerFab-L177AF&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;3.1 Expression of Herfab-L177AF&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-azido-L-phenylalanine (AF)</td><td>Bachem</td><td>F-3075.0001</td><td>1 g</td></tr><tr><td>L(+)-Arabinose, 99%</td><td>Acros</td><td>104981000</td><td>100 g</td></tr><tr><td>Hydrochloric acid, 35~37%</td><td>SAMCHUN</td><td>H0256</td><td>500 mL</td></tr><tr><td colspan="4">&lt;strong&gt;3.2 Cell Lysis&lt;/strong&gt;</td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99%</td><td>SAMCHUN</td><td>T1351</td><td>500 g</td></tr><tr><td>EDTA disodium salt dihydrate, 99.5%</td><td>SAMCHUN</td><td>E0064</td><td>1 kg</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td>500 g</td></tr><tr><td>Lysozyme</td><td>Siyaku</td><td>126-0671</td><td>1 g</td></tr><tr><td colspan="4">&lt;strong&gt;3.3 Ni-NTA Affinity Chromatography&lt;/strong&gt;</td></tr><tr><td>Ni-NTA resin</td><td>QIAGEN</td><td>30210</td><td>25 mL</td></tr><tr><td>Polypropylene column</td><td>QIAGEN</td><td>34924</td><td>50/pack, 1 mL capacity</td></tr><tr><td>Imidazole, 99%</td><td>SAMCHUN</td><td>I0578</td><td>1 kg</td></tr><tr><td>Sodium phosphate monobasic, 98%</td><td>SAMCHUN</td><td>S0919</td><td>1 kg</td></tr><tr><td>Sodium Chloride, 99%</td><td>SAMCHUN</td><td>S2907</td><td>1 kg</td></tr><tr><td colspan="4">&lt;strong&gt;4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-promoted Azide-alkyne Cycloaddition (SPAAC)&lt;/strong&gt;</td></tr><tr><td>Cy5.5-ADIBO&amp;nbsp;</td><td>FutureChem</td><td>FC-6119</td><td>1 mg</td></tr><tr><td colspan="4">&lt;strong&gt;5. Purification of Labeled HerFab&lt;/strong&gt;</td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>MILLIPORE</td><td>UFC500396</td><td>96/pack, 500 &amp;#956;L capacity</td></tr><tr><td colspan="4">&lt;strong&gt;6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning&lt;/strong&gt;</td></tr><tr><td>1,4-Dithio-DL-threitol, DTT, 99.5%</td><td>Sigma</td><td>10708984001</td><td>10 g</td></tr><tr><td>NuPAGE LDS Sample Buffer, 4x</td><td>Thermofisher</td><td>NP0007</td><td>10 mL</td></tr><tr><td>MES running buffer</td><td>Thermofisher</td><td>NP0002</td><td>500 mL</td></tr><tr><td>Nupage Novex 4-12% SDS PAGE gels</td><td>Thermofisher</td><td>NO0321</td><td>12-well</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Wako</td><td>031-17922</td><td>25 g</td></tr><tr><td>Typhoon 9210 variable mode imager</td><td>Amersham Biosciences</td><td></td><td></td></tr></tbody></table>

efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

In this study, an antibody fragment was site-specifically conjugated with a fluorophore by incorporating an azide-containing amino acid into the fragment and reacting the mutant antibody fragment with a strained cyclooctyne (Figure 1). HerFab was selected as the target antibody fragment into which AF was incorporated as an azide-containing amino acid. To choose the residue in HerFab for the replacement with AF, the X-ray crystal structure of HerFab was analyzed. 30 Important requirements for the residue include enough distance from the antibody binding site to minimize the decrease in its binding affinity and solvent accessibility for efficient conjugation reaction. Leucine at position 177 was a decent candidate because the residue is well exposed to outside of the antibody and located near the interface of two immunoglobulin domains, which is distant from the antibody binding site.
Initially, the expression plasmid for HerFab with a C-terminal His6-tag was constructed, and an amber codon was introduced at the position 177 for AF incorporation by site-directed mutagenesis. The mutant HerFab containing AF was expressed in the presence of 1 mM AF by co-expressing the evolved tRNATyr and aminoacyl-tRNA synthetase pair.27 Ni-NTA affinity purification was performed for the mutant proteins expressed in the presence and absence of AF, and the following SDS-PAGE analysis showed that the full-length antibody fragment was obtained only in the presence of AF (Figure 2). Next, the mutant antibody fragment containing AF was evaluated for conjugation reaction with a Cy5.5-linked aza-dibenzocyclooctyne derivative (Cy5.5-ADIBO). The antibody fragment and Cy5.5-ADIBO were reacted in a phosphate buffer for 6 h, and the reaction mixture was analyzed by SDS-PAGE in the presence (+) and absence (&#8722;) of DTT (Figure 3a).25 The reaction with a wild type antibody fragment was also carried out and analyzed as a control. The fluorescence images clearly showed conjugation of the mutant antibody fragment with Cy5.5-ADIBO, while no conjugation was observed in the reaction with the wild type fragment. Electron spray ionization mass spectrometric (ESI-MS) analysis showed quantitative conjugation with no detectable unconjugated fragment (Figure 3b). Overall, these results showed that the mutant HerFab containing AF can be efficiently and conveniently conjugated with strained cyclooctynes by SPAAC without any additional reagent.
<img alt="Figure 1" src="/files/ftp_upload/54922/54922fig1.jpg" /> 
Figure 1: Schematic illustration of site-specific antibody labeling by SPAAC. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54922/54922fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54922/54922fig2.jpg" /> 
Figure 2: Expression of mutant HerFab containing AF at position 177 (L177) in the presence of the evolved tRNA/aaRS pair. Expression of HerFab-L177AF in LB medium containing the evolved tRNA/AFRS pair and 1 mM AF. Purified samples were analyzed by SDS-PAGE in the presence (+) or absence (&#8722;) of DTT, and the gel was stained with a commercial protein stain. Figure adapted from Ko, W. et al.27. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54922/54922fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54922/54922fig3.jpg" /> 
Figure 3: Conjugation reaction of HerFab-L177AF with Cy5.5-ADIBO. (a) SDS-PAGE analysis of conjugation reaction of wild type HerFab and HerFab-L177AF with Cy5.5-ADIBO. The reaction mixtures were analyzed in the presence (+) and absence (&#8722;) of DTT, and protein bands were visualized by a commercial protein stain (left) and fluorescence imaging (right). (b) ESI-MS analyses of HerFab-L177AF (left) and HerFab-L177AF labeled with Cy5.5-ADIBO (right): expected mass difference between HerFab- L177AF and the conjugated antibody = 1,190 Da, observed mass difference = 1,190 Da. Figure adapted from Ko, W. et al.27. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54922/54922fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

efficient-site-specific-antibody-labeling-strain-promoted-azide

Nanyang Technological University

Research

JoVE Journal

Biochemistry

20.7K Views.  Sogang University. Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Video: Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Chemistry

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunology and Infection

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

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 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 ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

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