Name: Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition (Video) | JoVE Journal
Uploaded: 2016-12-23T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Learn site-specific antibody labeling using strain-promoted azide-alkyne cycloaddition for protein engineering. This method enables efficient probe incorporation into antibody fragments through simple mixing.

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5 minutes

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>

<ol><li>Wang, L., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+genetic+code%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Expanding the genetic code.</a> Angew. Chem. Int. Ed. 44 (1), 34-66 (2004).</li>
<li>Wu, X., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+at+the+interface+of+chemistry+and+biology%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Synthesis at the interface of chemistry and biology.</a> J. Am. Chem. Soc. 131 (35), 12497-12515 (2009).</li>
<li>Liu, C. C., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adding+new+chemistries+to+the+genetic+code%5BTitle%5D)+AND+%22Annu.+Rev.+Biochem%22%5BJournal%5D)">Adding new chemistries to the genetic code.</a> Annu. Rev. Biochem. 79, 413-444 (2010).</li>
<li>Wang, L., Zhang, Z., Brock, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Addition+of+the+keto+functional+groupto+the+genetic+code+of+Escherichia+coli%5BTitle%5D)+AND+%22proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Addition of the keto functional groupto the genetic code of Escherichia coli.</a> proc. Natl. Acad. Sci. U.S.A. 100 (1), 56-61 (2003).</li>
<li>Chin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Addition+of+p-azido-L-phenylalanine+to+the+genetic+code+of+Escherichia+coli%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.</a> J. Am. Chem. Soc. 124 (31), 9026-9027 (2002).</li>
<li>Deiters, A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+incorporation+of+an+alkyne+into+proteins+in+Esshcerichia+coli%5BTitle%5D)+AND+%22Bioorg.+Med.+Chem.+Lett%22%5BJournal%5D)">In vivo incorporation of an alkyne into proteins in Esshcerichia coli.</a> Bioorg. Med. Chem. Lett. 15 (5), 1521-1524 (2005).</li>
<li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+encoded+copper-free+click+chemistry%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Genetically encoded copper-free click chemistry.</a> Angew. Chem. Int. Ed. 50 (17), 3878-3881 (2011).</li>
<li>Seitchik, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+transcyclooctenes%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with transcyclooctenes.</a> J. Am. Chem. Soc. 134 (6), 2898-2901 (2014).</li>
<li>Lee, Y. -J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetically+encoded+acrylamide+fuctionality%5BTitle%5D)+AND+%22ACS+Chem.+Biol%22%5BJournal%5D)">A genetically encoded acrylamide fuctionality.</a> ACS Chem. Biol. 8 (8), 1664-1670 (2013).</li>
<li>Lang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+encoded+norbornene+directs+site-specific+cellular+protein+labelling+via+a+rapid+bioorthogonal+raction%5BTitle%5D)+AND+%22Nat+Chem%22%5BJournal%5D)">Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal raction.</a> Nat Chem. 4 (4), 298-304 (2012).</li>
<li>Lang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+encoding+of+bicyclononynes+and+trans-cyclooctenes+for+site-specific+protein+labeling+in+vitro+and+in+live+mammalian+cells+via+rapid+fluorogenic+Diels-Alder+reactions%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.</a> J. Am. Chem. Soc. 134 (25), 10317-10320 (2012).</li>
<li>Jewett, J. C., Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+Cu-free+click+chemistry+with+readily+synthesized+biarylazacyclooctynones%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones.</a> J. Am. Chem. Soc. 132 (11), 3688-3690 (2010).</li>
<li>Debets, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Azadibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+%283+%2B+2%29+cycloaddition%5BTitle%5D)+AND+%22Chem.+Commun%22%5BJournal%5D)">Azadibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition.</a> Chem. Commun. 46 (1), 97-99 (2010).</li>
<li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+mechanism+to+mouse%3A+a+tale+of+two+bioorthogonal+reactions%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">From mechanism to mouse: a tale of two bioorthogonal reactions.</a> Acc. Chem. Res. 44 (9), 666-676 (2011).</li>
<li>Debets, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioconjugation+with+strained+alkenes+and+alkynes%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Bioconjugation with strained alkenes and alkynes.</a> Acc. Chem. Res. 44 (9), 805-815 (2011).</li>
<li>Mbua, N. E., Guo, J., Wolfert, M. A., Steet, R., Boons, G. -J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain-promoted+alkyne%E2%88%92azide+cycloadditions+%28SPAAC%29+reveal+new+features+of+glycoconjugate+biosynthesis%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Strain-promoted alkyne−azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.</a> ChemBioChem. 12 (12), 1912-1921 (2011).</li>
<li>Kuzmin, A., Poloukhtine, A., Wolfert, M. A., Popik, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+functionalization+using+catalyst-free+azide-alkyne+cycloaddition%5BTitle%5D)+AND+%22Bioconjug.+Chem%22%5BJournal%5D)">Surface functionalization using catalyst-free azide-alkyne cycloaddition.</a> Bioconjug. Chem. 21 (11), 2076-2085 (2011).</li>
<li>Yao, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorophore+targeting+to+cellular+proteins+via+enzymemediated+azide+ligation+and+strain-promoted+cycloaddition%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition.</a> J. Am. Chem. Soc. 134 (8), 3720-3728 (2012).</li>
<li>Hao, Z., Hong, S., Chen, X., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introducing+bioorthogonal+functionalities+into+proteins+in+living+cells%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Introducing bioorthogonal functionalities into proteins in living cells.</a> Acc. Chem. Res. 44 (9), 742-751 (2011).</li>
<li>Reddington, S. C., Tippmann, E. M., Jones, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Residue+choice+defines+efficiency+and+influence+of+bioorthogonal+protein+modification+via+genetically+encoded+strain+promoted+Click+chemistry%5BTitle%5D)+AND+%22Chem.+Commun%22%5BJournal%5D)">Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.</a> Chem. Commun. 48 (9), 8419-8421 (2012).</li>
<li>Chari, R. V. J., Miller, M. L., Widdison, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody-drug+conjugates%3A+an+emerging+concept+in+cancer+therapy%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Antibody-drug conjugates: an emerging concept in cancer therapy.</a> Angew. Chem. Int. Ed. 53 (15), 3751-4005 (2014).</li>
<li>Tsao, M., Tian, F., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+staudinger+modification+of+proteins+containing+p-Azidophenylalanine%5BTitle%5D)+AND+%22ChemBioChem%22%5BJournal%5D)">Selective staudinger modification of proteins containing p-Azidophenylalanine.</a> ChemBioChem. 6 (12), 2147-2149 (2005).</li>
<li>Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+general+and+efficient+method+for+the+site-specific+dual-labeling+of+proteins+for+single+molecule+fluorescence+resonance+energy+transfer%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.</a> J. Am. Chem. Soc. 130 (52), 17664-17665 (2008).</li>
<li>Milles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Click+strategies+for+single-molecule+protein+fluorescence%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Click strategies for single-molecule protein fluorescence.</a> J. Am. Chem. Soc. 134 (11), 5187-5195 (2012).</li>
<li>Jang, S., Sachin, K., Lee, hJ., Kim, D. W., Lee, hS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis%5BTitle%5D)+AND+%22Bioconjug.+Chem%22%5BJournal%5D)">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjug. Chem. 23 (11), 2256-2261 (2012).</li>
<li>Kazane, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-assembled+antibody+multimers+through+peptide+nucleic+acid+conjugation%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Self-assembled antibody multimers through peptide nucleic acid conjugation.</a> J. Am. Chem. Soc. 135 (1), 340-346 (2012).</li>
<li>Ko, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+site-specific+antibody+labeling+by+strain-promoted+azide-alkyne+cycloaddition%5BTitle%5D)+AND+%22Bull.+Korean+Chem.+Soc%22%5BJournal%5D)">Efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition.</a> Bull. Korean Chem. Soc. 36 (9), 2352-2354 (2015).</li>
<li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> J. Mol. Biol. 395 (2), 361-374 (2010).</li>
<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li>
<li>Cho, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+extracellular+region+of+HER2+alone+and+in+complex+with+the+Herceptin+Fab%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.</a> Nature. 421 (6924), 756-760 (2003).</li>
<li>Kolb, H. C., Finn, M. G., Sharpless, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Click+chemistry+%3A+Diverse+chemical+function+from+a+few+good+reactions%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">Click chemistry : Diverse chemical function from a few good reactions.</a> Angew. Chem. Int. Ed. 40 (11), 2004-2021 (2001).</li>
</ol>

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 e...

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

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 <...

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

Research

JoVE Journal

Biochemistry

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.

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

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.

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

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

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.

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

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.

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

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.

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

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.

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

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.

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Learn site-specific antibody labeling using strain-promoted azide-alkyne cycloaddition for protein engineering. This method enables efficient probe incorporation into antibody fragments through simple mixing.

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

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