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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Catalyzed+Azide-Alkyne+Click+Chemistry+for+Bioconjugation.">Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation.</a> Current Protocols in Chemical Biology. 3 (4), 153-162 (2011).</li><li>Prasanna, M., 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=Semisynthetic+glycoconjugate+based+on+dual+role+protein/PsaA+as+a+pneumococcal+vaccine.">Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine.</a> European Journal of Pharmaceutical Sciences: Official Journal of the European Federation for Pharmaceutical Sciences. 129, 31-41 (2019).</li><li>Morrison, K. 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 t...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>E. coli str. B, F- ompT gal dcm lon hsdSB(rB-mB-) &#955;(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(&#955;S)</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 &#181;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 BamHI and XhoI restriction sites with a TEV protease sequence followed by a His6 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 tRNACUA 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 formulations<su...

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

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

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.