E.C. gratefully acknowledges the financial support from La R&#233;gion Pays de la Loire (Pari Scientifique Program &#34;BioSynProt&#34;), in particular a doctoral fellowship to T.V. We also acknowledge Dr Robert B. Quast (INRA UMR0792, CNRS UMR5504, LISBP, Toulouse, France) for his precious technical advices.

1. Synthesis of the UAA: propargyl-lysine (PrK)
<ol>
	<li>Synthesis of N&#945;-Boc-propargyl-lysine18
	<ol>
		<li>Dissolve 500 mg of Boc-L-Lys-OH (2.03 mmol) in a mixture of aqueous 1 M NaOH (5 mL) and THF (5 mL) in a flask and fit the flask with a silicon septum.</li>
		<li>Cool the flask in an ice bath and then add 158 &#181;L of propargyl chloroformate (1.62 mmol) dropwise (over a 2-3 min period) using a microsyringe while stirring.</li>
		<li>Warm the reaction mixture to room temperature and continue stirring for 10 h.</li>
		<li>Cool down solutions of 50 mL of diethyl ether, 50 mL of aqueous 1 M hydrochloric acid and 60 mL of ethyl acetate in an ice bath.</li>
		<li>Cool the crude reaction mixture in an ice bath and pour the mixture into a separation funnel. Extract the mixture with 50 mL of diethyl ether. Discard the organic layer.</li>
		<li>Cautiously add aqueous 1 M hydrochloric acid to the aqueous phase in the separation funnel. Then extract the aqueous layer twice using 30 mL of ethyl acetate. Verify the presence of N-Boc-propargyl-lysine in the organic phase by TLC using CH2Cl2-methanol (9:1) as eluent.</li>
		<li>Dry the combined organic layers over MgSO4, filter off the solid phase and concentrate the filtrate under reduced pressure on a rotary evaporator.</li>
		<li>Dissolve a sample of the crude oily N&#945;-Boc-propargyl-lysine in deuterated chloroform (CDCl3) and control its identity by 1H NMR. 
		CAUTION: Extraction may result in a buildup of pressure. Release any pressure buildup frequently.</li>
	</ol>
	</li>
	<li>Synthesis of the unnatural amino acid propargyl-L-lysine (PrK)
	<ol>
		<li>Introduce N&#945;-Boc-propargyl-lysine in a round bottom flask equipped with a septum.</li>
		<li>Add 4 mL of anhydrous dichloromethane (CH2Cl2) to the flask under argon to dissolve the N&#945;-Boc-propargyl-lysine.</li>
		<li>Add 4 mL of trifluoroacetic acid (TFA) dropwise using a syringe while stirring.</li>
		<li>Stir the reaction mixture for 1 h at RT. Monitor the reaction by TLC using CH2Cl2-methanol (9:1) as eluent.</li>
		<li>Concentrate the reaction mixture under reduced pressure.</li>
		<li>Add diethyl ether to the crude residue and incubate it at 4 &#176;C for 1 h to precipitate the PrK. When working on higher scale, if the PrK is not completely precipitated, triturate to precipitate it and extend the incubation time if needed.</li>
		<li>Filter the PrK in the form of a white solid on a fritted-glass.</li>
		<li>Dissolve an aliquot of the PrK in D2O. Then carry out NMR analyses to control its identity and purity.</li>
		<li>For further use, dissolve the unnatural amino acid PrK in distilled water at a final concentration of 100 mM and store at -20 &#176;C as 1 mL aliquots.</li>
	</ol>
	</li>
</ol>
2. Production of the recombinant protein modified by PrK
<ol>
	<li>Plasmid preparation
	<ol>
		<li>Construct an expression plasmid (pET24d-mPsaAK32TAG-ENLYFQ-HHHHHH) that contains the target mature Pneumococcal surface adhesin A (mPsaA) gene (pET24d-mPsaA-WT) followed by a Tobacco Etch Virus (TEV) protease sequence by cloning the insert between the BamHI and XhoI restriction sites of the pET24d plasmid. This will introduce a His6 tag at the C-terminus of the protein. Replace the codon of lysine-32 with the amber codon (TAG), using conventional site-directed mutagenesis technique.</li>
		<li>Construct a second expression plasmid (pEVOL-MmPylRS) containing two copies of the gene coding for the pyrrolysyl-tRNA synthetase from Methanosarcina mazei (MmPylRS) and the gene coding for the corresponding tRNAPyr as previously described19. Use this specially designed plasmid vector, pEVOL, for efficient incorporation of UAAs. 
		NOTE: The detailed plasmids information is described in Supplemental File 1.</li>
	</ol>
	</li>
	<li>Co-transformation of plasmids into the expression strain
	<ol>
		<li>Thaw a 100 &#181;L aliquot of chemically competent Escherichia coli BL21(DE3) on ice for 5 min.</li>
		<li>Add 1 &#181;L of each plasmid (50-100 ng of each) into the cells and incubate for 30 min on ice.</li>
		<li>Transfer the 1.5 mL microtube with the thawed competent cells in an incubator at 42 &#176;C for 45 s and then move it back to ice for 2 min.</li>
		<li>Add 900 &#181;L of LB medium and incubate under shaking for 1 h at 37 &#176;C to allow antibiotic expression. Then plate the bacteria onto LB agar with 25 &#181;g/mL of kanamycin and 30 &#181;g/mL of chloramphenicol. Allow bacteria growth overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Expression of proteins modified with PrK
	<ol>
		<li>Inoculate a single co-transformed colony in 5 mL of LB medium with antibiotics (25 &#181;g/mL of kanamycin and 30 &#181;g/mL of chloramphenicol). Incubate overnight at 37 &#176;C with shaking.</li>
		<li>Dilute the primary culture (5 mL) into 500 mL of auto-induction medium containing antibiotics, 0.02% of L-arabinose and 1 mM of the unnatural amino acid PrK and incubate it at 37 &#176;C for 24 h with shaking. Include a negative control by performing the culture without PrK in parallel and a positive control by performing the culture of a clone containing the wt protein.</li>
		<li>Aliquot 5 mL out of the 500 mL culture medium and centrifuge for 10 min at 5,000 x g. Discard the supernatant and freeze the pellet at -20 &#176;C. Harvest cells from the remaining 495 mL by centrifugation for 10 min at 5,000 x g. Discard the supernatant and freeze the pellet at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Analyze crude cell extracts from the 5 mL culture samples by SDS-PAGE and western Blot analysis
	<ol>
		<li>Resuspend 5 mL cell pellets into 250 &#181;L of lysis buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 8, 5 mM imidazole, 0.2 mM PMSF) and transfer it into a 1.5 mL microtube.</li>
		<li>Lyse cells by freezing the tubes in liquid nitrogen, thawing it in a 42 &#176;C bath and vortexing at high speed for 30 s. Repeat this step 3 times.</li>
		<li>Centrifuge samples at 17,000 x g for 10 min to eliminate cell debris.</li>
		<li>Take 10 &#181;L of the supernatant and add 5 &#181;L of water and 5 &#181;L of loading buffer (bromophenol blue, SDS, &#946;-mercaptoethanol). Heat the samples for 5 min at 100 &#176;C and carry out SDS-PAGE and western Blot analyses.</li>
	</ol>
	</li>
	<li>Protein purification by gravity flow-bench affinity chromatography using Nickel-NTA beads
	<ol>
		<li>Resuspend the cell pellets (from the 495 mL culture) into 20 mL of lysis buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 8, 5 mM imidazole, 0.2 mM PMSF).</li>
		<li>Add 5 &#181;L of DNase I (1 mg/mL) and 500 &#181;L of lysozyme (50 mg/mL) into the suspension and allow lysis by incubating the suspension at 37 &#176;C during 30 min.</li>
		<li>Sonicate the cells during 5 min (cycles of 5 s-5 s, amplitude 50%) and then remove the cell debris by centrifugation at 20,000 x g for 30 min followed by filtration on 0.45 &#181;m filter.</li>
		<li>Add Ni-NTA resin to the suspension (500 &#181;L for 500 mL of cell culture) and mix gently at 4 &#176;C for 1 h.</li>
		<li>Pour the suspension into a polypropylene column and collect the unbound fraction.</li>
		<li>Wash the resin with 10 mL of washing buffer containing 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 10 mM imidazole. Wash the resin a second time with 5 mL of washing buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 20 mM imidazole). Collect the wash fractions.</li>
		<li>Elute the his-tagged protein with 1 mL of elution buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 300 mM imidazole). Repeat this step 4 times and collect all the elution fractions.</li>
		<li>Analyze the crude lysate as well as the 7 purification fractions by SDS-PAGE on a 12% acrylamide gel.</li>
		<li>Combine the fractions containing pure His-tagged protein and dialyze it against 1 L of TEV protease buffer (50 mM Tris-HCl, 0.5 mM EDTA, pH 8) overnight by using a dialysis membrane (cut-off MW 6000-8000 Da). Measure the concentration of the protein at 280 nm with a molar extinction coefficient of 37 360 cm-1&#8729;M-1 and a molecular weight of 34.14 kDa for mPsaA.</li>
	</ol>
	</li>
</ol>
3. Removal of the histidine tag by TEV protease digestion
<ol>
	<li>Collect the protein sample into a 50 mL tube and add TEV buffer (50 mM Tris HCl, 0.5 mM EDTA, pH 8) up to 1 mL at a concentration of 2 mg/mL. 
	NOTE: The concentration may vary according to previous results. Protein concentrations that we have tested are in a typical 2-3 mg/mL range.</li>
	<li>Add 100 &#181;L of TEV protease (add 1 &#181;L containing 10 units of TEV protease for 20 &#181;g of protein to digest).</li>
	<li>Add 50 &#181;L of 0.1 M dithiothreitol (DTT).</li>
	<li>Complete with TEV buffer (50 mM Tris HCl, 0.5 mM EDTA, pH 8) up to 5 mL.</li>
	<li>Incubate overnight at 4 &#176;C with slow shaking. 
	NOTE: If digestion is not complete, add more TEV protease, incubate for longer time or at higher temperature up to 30 &#176;C.</li>
	<li>Dialyze the digested protein to remove EDTA at 4 &#176;C overnight by using a dialysis membrane (cut-off 6000-8000 Da) against phosphate buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 5 mM imidazole).</li>
	<li>To eliminate the TEV protease and the undigested protein, incubate the mix with Ni-NTA beads and mix gently for 1 h at 4 &#176;C.</li>
	<li>Pour the suspension into a polypropylene column. Collect the unbound fraction and wash the column with 5 mL of washing buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 10 mM imidazole) 
	NOTE: The protein of interest should be recovered in the unbound and washing fractions.</li>
	<li>Elute the TEV protease and the undigested protein by adding 5 mL of elution buffer (50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 300 mM imidazole) on the column. Check the fractions for protein contents at 280 nm and by SDS-PAGE analysis.</li>
	<li>Check the efficiency of the digestion by loading digested samples on an SDS PAGE with the undigested protein as a control.</li>
	<li>Dialyze the digested protein against 1 L of click buffer (50 mM Na2HPO4/NaH2PO4, pH8) at 4 &#176;C overnight with a dialysis membrane (cut-off 6000-8000 Da) to remove imidazole as well as to exchange the buffer, and measure the concentration of the protein at 280 nm with molar extinction coefficient and molecular weight of mPsaA (37 360 cm-1&#8729;M-1 and MW 34.14 kDa).</li>
</ol>
4. Assessment of the unnatural amino acid propargyl-lysine accessibility and functionality for click chemistry
NOTE: Conjugate the mPsaA with 6-hexachloro-fluorescein-azide using the protocol described by Presolski et al.20 for click chemistry.
<ol>
	<li>Take 432.5 &#181;L of PrK-mutated protein at a concentration of 57.8 &#181;M into a 2 mL microtube. 
	NOTE: A minimum concentration of 2 &#181;M of alkyne is acceptable. If the protein concentration is lower, concentrate it with a centrifugal concentrator or favor the balance of the reaction by increasing azide/alkyne molar ratio.</li>
	<li>Add 10 &#181;L of 5 mM 6-hexachloro-fluorescein-azide and then add a premix of 2.5 &#181;L of CuSO4 solution at 20 mM and 7.5 &#181;L of Tris(benzyltriazolylmethyl)amine (THPTA) at 50 mM (stock solutions concentrations).
	<ol>
		<li>Add 25 &#181;L of aqueous 100 mM aminoguanidine hydrochloride.</li>
		<li>Add 25 &#181;L of 20 mg/mL an extemporaneously prepared aqueous solution of sodium ascorbate.</li>
		<li>Close the tube, mix by inverting several times and incubate at room temperature for 2 h.</li>
		<li>Stop the reaction by adding 50 &#181;L of 0.5 M EDTA.</li>
		<li>Take 15 &#181;L of the reaction mixture and put it a microtube, add 5 &#181;L of loading buffer (bromophenol blue, SDS, &#946;-mercaptoethanol), heat the mixture at 100 &#176;C for 5 min, and then load it into a 12% acrylamide gel. After migration, visualize the fluorescent conjugate on the gel under UV light at 312 nm.</li>
	</ol>
	</li>
</ol>
5. Conjugation of mPsaA with an azido-functionalized carbohydrate antigen (Pn14TS-N3) by click chemistry
<ol>
	<li>Coupling
	<ol>
		<li>Take 432.5 &#181;L of PrK-mutated protein at 57.8 &#181;M into a 2 mL microtube.</li>
		<li>Add 10 &#181;L of 5 mM Pn14TS-N321 in water then add a premix of 2.5 &#181;L of CuSO4 solution at 20 mM and 7.5 &#181;L of THPTA at 50 mM. 
		NOTE: Synthesis of Pn14TS-N3, a tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide, has been described in reference 21. Theoretically, any carbohydrate antigen containing an azide function can be used.</li>
		<li>Add 25 &#181;L of 100 mM aminoguanidine hydrochloride.</li>
		<li>Add 25 &#181;L of 20 mg/mL extemporaneously prepared aqueous solution of sodium ascorbate.</li>
		<li>Close the tube, mix by inverting several times and incubate at RT during 2 h.</li>
		<li>Stop the reaction by adding 50 &#181;L of 0.5 M EDTA.</li>
		<li>Take 15 &#181;L of samples and analyze by SDS-PAGE.</li>
	</ol>
	</li>
	<li>Gel filtration purification of the glycoconjugate
	<ol>
		<li>Purify the glycoconjugate by applying it to a steric exclusion agarose column (15 x 600 bed dimensions, 3,000-70,000 fractionation range), equilibrated with 100 mM PBS buffer, pH 7.3 at a 0.8 mL/min flow with detection at 280 nm.</li>
		<li>Collect the fractions containing the glycoconjugate. 
		NOTE: For prolonged storage, dialyze the glycoconjugate against 1 L of H2O twice for 2 h and then overnight at 4 &#176;C by using dialysis membrane (cut-off Mw 6000-8000 Da), then freeze-dry and store the glycoconjugate at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+psaA+in+all+90+serotypes+of+Streptococcus+pneumoniae+by+PCR+and+potential+of+this+assay+for+identification+and+diagnosis.">Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis.</a> Journal of Clinical Microbiology. 38 (1), 434-437 (2000).</li><li>Lin, H., Lin, Z., Meng, C., Huang, J., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+immunogenicity+of+capsular+polysaccharide-surface+adhesin+A+(PsaA)+conjugate+of+Streptococcuspneumoniae.">Preparation and immunogenicity of capsular polysaccharide-surface adhesin A (PsaA) conjugate of Streptococcuspneumoniae.</a> Immunobiology. 215 (7), 545-550 (2010).</li><li>Safari, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+smallest+structure+capable+of+evoking+opsonophagocytic+antibodies+against+Streptococcus+pneumoniae+type+14.">Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14.</a> Infection and Immunity. 76 (10), 4615-4623 (2008).</li><li>Wang, Q., Parrish, A. R., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code+for+biological+studies.">Expanding the genetic code for biological studies.</a> Chemistry &amp; Biology. 16 (3), 323-336 (2009).</li><li>Lawrence, M. C., Pilling, P. A., Epa, V. C., Berry, A. M., Ogunniyi, A. D., Paton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+pneumococcal+surface+antigen+PsaA+reveals+a+metal-binding+site+and+a+novel+structure+for+a+putative+ABC-type+binding+protein.">The crystal structure of pneumococcal surface antigen PsaA reveals a metal-binding site and a novel structure for a putative ABC-type binding protein.</a> Structure (London, England: 1993). 6 (12), 1553-1561 (1998).</li><li>Wright, T. H., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+mutagenesis+for+installation+of+natural+and+unnatural+amino+acid+side+chains+into+recombinant+proteins.">Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins.</a> Nature Protocols. 12 (10), 2243-2250 (2017).</li><li>Dadov&#225;, J., Galan, S. R., Davis, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+modified+proteins+via+functionalization+of+dehydroalanine.">Synthesis of modified proteins via functionalization of dehydroalanine.</a> Current Opinion in Chemical Biology. 46, 71-81 (2018).</li><li>Worst, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residue-specific+Incorporation+of+Noncanonical+Amino+Acids+into+Model+Proteins+Using+an+Escherichia+coli+Cell-free+Transcription-translation+System.">Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.</a> Journal of Visualized Experiments. (114), (2016).</li><li>Carboni, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GBS+type+III+oligosaccharides+containing+a+minimal+protective+epitope+can+be+turned+into+effective+vaccines+by+multivalent+presentation.">GBS type III oligosaccharides containing a minimal protective epitope can be turned into effective vaccines by multivalent presentation.</a> The Journal of Infectious Diseases. , (2019).</li></ol>

The authors have nothing to disclose.

Site-directed mutagenesis is a straightforward strategy to incorporate specific amino acids at a defined position of a protein which remains barely used with the aim of preparing glycoconjugate vaccines7,8,14. Classical mutagenesis based on the 20 natural amino acids approach is highly efficient since no modification of the translation machinery is required. Cysteine mutations are usually targeted to further explore the unique thiol reactivity either directly or in two steps (e.g., after its modification into a dehydroalanine intermediate, a strategy called post-translational mutagenesis)27,28. Genetic code expansion is perhaps even more attractive and flexible since it allows the direct incorporation of a wide range of UAAs with diverse functionalities9,10. While several UAAs can be incorporated simultaneously within a protein29, the number of mutations is usually more limited. We herein applied the related amber stop codon strategy to introduce a single propargyl-lysine in a carrier protein. The incorporation can take place at any position provided the sidechain of the initial amino acid was surface-exposed, a criterion easily determined from X-ray crystallographic structures or in silico modeling. Moreover, it is not limited to propargyl-lysine but can be extended to any UAA functionalized with a biorthogonal function which will later serve as an anchor to graft the incoming carbohydrate antigen and for which an orthogonal aaRS/tRNA pair exists.
One of the drawbacks of the strategy is the possible production of truncated protein, resulting from the release of the peptidyl sidechain when reading the amber stop codon, as a side-product. Even if we did not observe any truncated form here (probably degraded by the bacteria because of it very small size), a histidine tag has been added at C-terminus of the protein to facilitate the purification of the expected full-length mutated protein from impurities and noticeably from the truncated protein (which by essence does not express the histidine tag sequence). This can become essential if the UAA incorporation is carried out near the protein C-terminus since purification cannot be attempted using alternative chromatography techniques like gel filtration.
For most applications removal of the histidine tag is not mandatory. However, it may be useful regarding the design of glycoconjugate vaccine as part of the immune system may be diverted against the tag sequence. For this proof-of-concept, we inserted an amino acid length sequence specifically cleaved by the TEV protease which leaves five extra amino acids on the carrier protein after digestion.
The conjugation step between the alkyne of the propargyl-lysine and a representative synthetic oligosaccharide Pn14TS related to a pneumococcal capsule and bearing a complementary azide was carried out according to a click chemistry protocol reported by Presolski et al.20 If necessary, completion of the reaction can easily be reached by increasing the reaction time or by modifying the ratio between the alkyne, azide and copper reactants and reagents. Copper salts are eliminated by treatment with excess EDTA followed by a short purification by steric exclusion chromatography.
The glycoconjugate obtained with the technique described in the present work can then be used to immunize mice. Having such fully-defined and easily modulated glycoconjugate in hands provides invaluable tools to evaluate the impact of the hapten/protein carrier connectivity on the immune response8. Since increasing the hapten/protein ratio is often correlated with enhanced anti-hapten humoral response when using short haptens30, one might be interested in testing conjugates with multiple haptens. The incorporation of multiple UAAs however needs some adjustments of the protocol as the incorporation of an UAA in the protein tends to decrease the yield of protein production due to the RF1 activity.
In definitive, this method is a powerful tool to gain access to homogeneous glycoconjugate vaccines facilitating their physico-chemical characterization and further carbohydrate antigen/carrier connectivity-immunogenicity relationship studies.

Glycoconjugate vaccines are essential elements of the vaccine arsenal available for the prophylactic treatment of infectious diseases. They are safe, well-tolerated and efficient in a broad age group including young infants. They provide the optimal defense against infections caused by capsulated bacteria like meningococcus, pneumococcus or Haemophilus influenzae type b1. Glycococonjugate vaccines are made of purified bacterial polysaccharides that form the capsules of bacteria or synthetic oligosaccharides that mimic these surface-expressed polysaccharides2, which are covalently linked to a carrier protein. The presence of a carrier protein is essential to promote protective humoral immune responses directed against the antigenic determinant expressed by the carbohydrate antigens3. Apart from a careful selection and production of the carbohydrate antigen, the features known to exert an influence on the efficacy of a glycoconjugate vaccine are: the nature of the carrier protein, the conjugation chemistry (including the nature and the length of the linker if used), or the saccharide/protein ratio3. Obviously, the positions at which the saccharide is conjugated to the protein as well as the number of connectivity points are relevant for immunogenicity. To date, these two parameters have hardly been studied because the preparation of the glycoconjugates remains largely empirical. Their synthesis usually relies on the use of amine or carboxylic acid functions of, respectively, lysine or aspartic/glutamic acid side-chain residues present on the carrier protein sequence. This leads not to a single but to a heterogeneous mixture of glycoconjugates.
Playing on the reactivity, accessibility or distribution of the amino acid residues in the protein gives rise to more defined glycoconjugates that are more reliable to document the effect of saccharide/protein connectivity4. A step forward towards this goal can be achieved by applying protein glycan coupling technology, a recombinant process that allows the production of controlled glycoconjugate vaccines in cell factories5,6. However, the glycosylation exclusively takes place at an asparagine residue within D/EXNYS/T sequons (whereby X is any out of the 20 natural amino acids), not naturally present on the carrier proteins.
Site selective mutagenesis and in particular incorporation of cysteines to exploit their highly and selective reactivity appears as an alternative7,8. Production of carrier proteins incorporating UAAs in their sequence can offer even more flexibility for homogeneous glycoconjugate vaccine preparation. More than 100 UAAs have been developed and further incorporated into various proteins9,10. Many of them contain bioorthogonal functions usually used to carry out post translational modifications11 or to graft biophysical probes12 or drugs13 but which are ideal handles for further conjugation with carbohydrate antigens. Successful examples have been claimed by Biotech14 using cell-free protein synthesis15 but preparation of glycoconjugate vaccines according to this strategy still waits for becoming popularized.
Application of the in vivo strategy for the production of mutated carrier protein needs a modified translational machinery that includes a specific codon, a tRNA recognizing the codon and an aminoacyl-tRNA synthetase (aaRS) which specifically catalyzes the transfer of the UAA on the tRNA (Figure 1)16. The pyrrolysine amber stop codon suppression is one of the most widely used methods to incorporate UAA, in particular the propargyl-lysine (PrK)17. The latter can in turn react with azido-functionalized carbohydrate haptens to provide fully defined, homogeneous glycococonjugates. In the present manuscript we describe how to synthesize the propargyl-L-lysine, an UAA carrying an alkyne handle, how to incorporate it into a target protein during its translation in a bacteria and finally how to perform conjugation between the modified protein and a hapten carrying an azide function using click chemistry.

<table><tbody><tr><td>AIM (autoinductif medium)</td><td>Formedium</td><td>AIMLB0210</td><td>Solid powder</td></tr><tr><td>Boc-Lys-OH</td><td>Alfa-Aesar</td><td>H63859</td><td>Solid powder</td></tr><tr><td>BL21(DE3)</td><td>Merck Novagen</td><td>69450</td><td>&lt;em&gt;E. coli&lt;/em&gt; str. B, F&lt;sup&gt;-&lt;/sup&gt; &lt;em&gt;ompT gal dcm lon hsdS&lt;sub&gt;B&lt;/sub&gt;&lt;/em&gt;(&lt;em&gt;r&lt;sub&gt;B&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;m&lt;sub&gt;B&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;&lt;/em&gt;) &amp;lambda;(DE3 [&lt;em&gt;lacI lacUV5-T7p07 ind1 sam7 nin5&lt;/em&gt;]) [&lt;em&gt;malB&lt;sup&gt;+&lt;/sup&gt;&lt;/em&gt;]&lt;sub&gt;K-12&lt;/sub&gt;(&amp;lambda;&lt;sup&gt;S&lt;/sup&gt;)</td></tr><tr><td>Dialysis membrane</td><td></td><td></td><td></td></tr><tr><td>DNAseI</td><td></td><td></td><td></td></tr><tr><td>Filter 0.45 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>L-arabinose</td><td></td><td></td><td></td></tr><tr><td>lysozyme</td><td></td><td></td><td></td></tr><tr><td>Ni-NTA resin</td><td>Machery Nagel</td><td>Protino</td><td>Ni-NTA beads in suspension into 20% ethanol</td></tr><tr><td>Pall centrifugal device</td><td></td><td></td><td></td></tr><tr><td>pET24d-mPsaAK32TAG-ENLYFQ-HHHHHH</td><td>this study</td><td></td><td>same as pET24d-mPsaA-WT but with a K32TAG mutation in the mPsaA gene</td></tr><tr><td>pET24d-mPsaA-WT</td><td>this study</td><td></td><td>pET24d plasmide with the Wt mPsaA gene cloned between the &lt;em&gt;BamHI&lt;/em&gt; and &lt;em&gt;XhoI&lt;/em&gt; restriction sites with a TEV protease sequence followed by a His&lt;sub&gt;6&lt;/sub&gt; tag at the C-terminal end of mPsaA gene and carrying the Kanamycine resistance gene</td></tr><tr><td>pEVOL plasmid</td><td>gift fromEdward Lemke EMBL (ref 19)</td><td></td><td>plasmide with p15A origin, two copies of MmPylRS (one under GlnS promoter and one under pAra promoter), one copy of the tRNA&lt;sup&gt;CUA&lt;/sup&gt; under the ProK promoter, the chloramphenicol resistance gene</td></tr><tr><td>Propargyl chloroformate</td><td>Sigma-Aldrich</td><td>460923</td><td>Liquid</td></tr><tr><td>Sonicator</td><td>Thermo Fisher</td><td>FB120-220</td><td></td></tr></tbody></table>

homogeneous glycoconjugate produced by combined unnatural amino acid incorporation and click-chemistry for vaccine purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

In this project, a homogeneous glycoconjugate vaccine was prepared using the amber stop codon suppression strategy to introduce an UAA at a defined site (Figure 1). Pneumoccocal surface adhesin A was selected as the carrier protein moiety. This protein is highly conserved and expressed by all strains of Streptococcus pneumoniae22. It is highly immunogenic and previously used as a carrier in pneumococcal vaccine formulations21,23. As a proof-of-concept, the UAA propargyl-lysine efficiently charged by the wild type pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair of the archaea Methanosarcina mazei was investigated. The propargyl-lysine is commercially available but can be advantageously prepared from Boc-L-lysine in only two synthetic steps (Figure 2). An amber codon was generated at a desired position in a pET24d plasmid containing the mPsaA gene. This plasmid was co-transformed with a pEVOL plasmid (a kind gift from Edward Lemke (EMBL19) containing orthogonal tools necessary to incorporate the propargyl-lysine, into competent E. coli BL21(DE3) strain. Positive co-transformed clones were selected using 25 &#181;g/mL kanamycin and 30 &#181;g/mL chloramphenicol. The plasmid pEVOL contains originally not one but two copies of the gene coding for MmPylRS to incorporate the propargyl-lysine residue: the first copy is under the control of a constitutive promoter while the expression of the other one is inducible in the presence of arabinose. However, we have noticed no dramatic decrease of propargyl-lysine incorporation if the MmPylRS gene under the control of constitutive promoter is suppressed.
The propargyl-lysine was introduced at position 32 in replacement of a lysine near the N-terminus of the PsaA. Any residue with a surface-exposed side-chain can virtually be exchanged in view of carrying out further conjugation. The mutated protein was produced in its mature form (mPsaAK32PrK) with inclusion of a cleavable 6-histidine tag sequence at its C-terminus. The efficiency of the mPsaAK32PrK production was checked by SDS-PAGE and Western Blot analysis using an anti-Histidine tag antibody, when growth was performed in the presence or the absence of the UAA propargyl-lysine and in comparison with the production of the wild type mPsaA (Figure 3). Visualization revealed a protein band at an expected molecular weight (Lanes 4, Figure 3A &#38; 3B). The presence of a full-length protein strongly indicates the successful incorporation of the PrK into mPsaA. The intensity is, however, lower than that observed for wild type mPsaA (Lanes 2, Figure 3A &#38; 3B). Leakage (i.e., production of the full-length protein without incorporation of the UAA) and premature release of the protein by the Release Factor RF1 during translation are two main drawbacks frequently encountered during this process. On one hand, no band at the expected molecular weight is visualized in the absence of propargyl-lysine meaning that no leakage occurred (Lanes 3, Figure 3A &#38; 3B) and indirectly confirmed that the band observed on Lanes 4 corresponds to mPsaAK32PrK. On the other hand, no band can be seen at low molecular weight that could correspond to the truncated form of mPsaA (Lane 4 on Figure 3A). The mPsaAK32PrK was then purified by affinity chromatography, with a typical yield of 8 mg/L (in comparison to 12-20 mg/L for the wild type protein) and the incorporation of the propargyl-lysine residue was finally confirmed by mass spectrometry (Figure 4). The histidine tag was removed upon proteolytic cleavage using TEV protease (Figure 4). The stability of the mPsaAK32PrK thus obtained was assessed by circular dichroism, which showed that the structure of the protein was not disturbed by the mutation of the Lysine 32 into a propargyl-lysine (data not shown).
Having the mPsaAK32PrK, the reactivity of the alkyne for click chemistry was assessed using an azido-functionalized fluorescein and further used to conjugate a synthetic oligosaccharide antigen &#946;-2-azidoethyl d-Galp-(1&#8594;4)-&#946;-d-Glcp-(1&#8594;6)-[&#946;-d-Galp-(1&#8594;4)]-&#946;-d-GlcpNAc (Pn14TS) (Figure 5). This tetrasaccharide is related to the S. pneumoniae type 14 capsular polysaccharide and has previously been conjugated to mPsaA using different conjugation chemistries8,21,24. Experiments here were done in comparison with wild type mPsaA as a control. The histidine tag was first removed upon proteolytic cleavage using the TEV protease. The digested mPsaAK32PrK and mPsaA WT were then conjugated to the fluoroprobe (Figure 6A) or Pn14TS (Figure 6B). The reaction was assessed by SDS-PAGE. The small increase in the molecular weight of the sample between lane 6 and 7 (Figure 6B) indicates a successful conjugation with the tetrasaccharide Pn14TS. Finally, the glycoconjugate was purified by gel filtration and its identity confirmed by mass spectrometry (Figure 6C). The conjugation by click chemistry being quantitative the majority of the mPsaAK32PrK was conjugated with the Pn14TS-N3 as illustrated by the mass spectrometry results (Figure 6C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60821/60821fig1.jpg" /> 
Figure 1: Incorporation of propargyl-lysine (PrK) into mPsaA during translation using an orthogonal pyrrolysyl-tRNA synthetase/tRNA pair and TAG codon reassignment25. During translation, endogenous synthetases catalyze the link between amino acids and corresponding tRNAs. Then, loaded tRNAs are used by the ribosomal machinery to generate the neo-synthesized polypeptide. According to the amber stop codon suppression strategy, an orthogonal aminoacyl-tRNA synthetase (aaRS) (herein a pyrrolysyl-tRNA synthetase from M. mazei), loads an UAA (herein PrK) on its cognate tRNA which designed anticodon can read the amber stop codon (TAG) on the mRNA. This specific recognition directs the incorporation of the UAA into the specific site on the target protein. Figure reproduced from Wang et al.25. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60821/60821fig2.jpg" /> 
Figure 2: Propargyl-lysine synthesis. (A) Steps of propargyl-lysine synthesis. Insert: Monitoring of the deprotection of Boc-l-Lys(prop-2-ynyloxycarbonyl)-OH intermediate: thin-layer chromatography on 0.25 mm silica gel plates with fluorescent indicator (GF254) and visualised by charring with vanillin in sulfuric acid/ethanol (1.5:95 v/v); eluent: CH2Cl2/MeOH (9:1), left lane: Boc-l-Lys(prop-2-ynyloxycarbonyl)-OH (Rf 0.90), right lane: crude propargyl-lysine, (Rf 0.38). 400 MHz 1H (B) and 13C NMR spectra (C) of propargyl-lysine recorded in D2O. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60821/60821fig3.jpg" /> 
Figure 3: Analysis of crude cell samples. (A) SDS-PAGE analysis and (B) Western blot analysis on crude cell samples. Lane 1: unstained protein marker; Lane 2: crude cell extract of wild type mPsaA; Lane 3: crude cell extract of mPsaAK32TAG grown in the absence of PrK; Lane 4: crude cell extraction of mPsaAK32TAG grown in the presence of PrK. Conditions: 12% acrylamide gel, running at 100 V, 2 h. SDS-PAGE stained by Coomassie blue; Western Blot revealed using anti-histidine tag antibody and secondary antibody coupled with AlexaFluor680. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60821/60821fig4.jpg" /> 
Figure 4: Histidine tag removal and mass spectrometry analysis. (A) SDS-PAGE analysis. Lane 1: unstained protein marker; Lane 2: crude cell extract; Lane 3: unbound fraction; Lane 4: wash fraction with 10 mM imidazole; Lane 5: wash fraction with 20 mM imidazole; Conditions: 12% acrylamide gel running at 100 V for 2 h, and stained by Coomassie blue; (B) MALDI-TOF-MS spectra of (top) mPsaA WT, theoretical MW 33 103 Da, found 33 106&#160;Da and (bottom) mPsaA K32PrK, theoretical 33 184 Da, found 33 192 Da. The found masses are within the expected margin error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60821/60821fig5.jpg" /> 
Figure 5: Schematic representation of the conjugation strategy by click chemistry. A single tetrasaccharide bearing an azide is specifically coupled to its complementary biorthogonal alkyne group on mPsaA K32PrK (mPsaA representation based on the 1PSZ PDB file, with a resolution of 2.0 &#197;26). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60821/60821fig6.jpg" /> 
Figure 6: Histidine-tag digestion and conjugation of mPsaA with (A) fluorescein-N3 and with (B) Pn14TS. (A) SDS-PAGE Lanes 1-3: WT mPsaA; Lane 4-6: mPsaAK32PrK; (B) Lane 1: unstained protein marker; Lane 2-4: WT mPsaA; Lane 5-7: mPsaAK32PrK. 2 &#181;g protein sample/lane, 12% acrylamide, 100 V, 2 h; (C) MALDI-TOF-MS spectra of the Pn14TS-mPsaAK32PrK theoretical MW 34 091 Da, found 34 088 Da. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/60821fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60821/pET-24-d_mPsaA_K32.pdf" target="_blank">Please click here to view this file (Right click to download).</a>

Watch this Scientific Journal Video about Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes at JoVE.com

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

homogeneous-glycoconjugate-produced-combined-unnatural-amino-acid

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4.9K Views.  Université de Nantes, CNRS, Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 6286. Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Video: Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

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).

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 ...

Biochemistry

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

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

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Chemistry

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 ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Summary

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Chapters in this video

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

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

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Generation of Monoclonal Antibodies Against Natural Products

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain