Name: homogeneous glycoconjugate produced by combined unnatural amino acid incorporation and click-chemistry for vaccine purposes
Uploaded: 2020-12-19T13:00:00
Description: Watch this Scientific Journal Video about Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes at JoVE.com

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 ...

<ol>
	<li>Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoconjugate+vaccines:+Principles+and+mechanisms.">Glycoconjugate vaccines: Principles and mechanisms.</a> Science Translational Medicine. 10 (456), (2018).</li><li>Verez-Bencomo, V., 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=A+synthetic+conjugate+polysaccharide+vaccine+against+Haemophilus+influenzae+type+b.">A synthetic conjugate polysaccharide vaccine against Haemophilus influenzae type b.</a> Science (New York, N.Y). 305 (5683), 522-525 (2004).</li><li>Berti, F., Adamo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+glycoconjugate+vaccines:+an+overview+of+classic+and+modern+approaches+for+protein+modification.">Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification.</a> Chemical Society Reviews. 47 (24), 9015-9025 (2018).</li><li>Stefanetti, 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=Sugar-Protein+Connectivity+Impacts+on+the+Immunogenicity+of+Site-Selective+Salmonella+O-Antigen+Glycoconjugate+Vaccines.">Sugar-Protein Connectivity Impacts on the Immunogenicity of Site-Selective Salmonella O-Antigen Glycoconjugate Vaccines.</a> Angewandte Chemie (International Ed. in English). 54 (45), 13198-13203 (2015).</li><li>Kay, E., Cuccui, J., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+production+of+recombinant+glycoconjugate+vaccines.">Recent advances in the production of recombinant glycoconjugate vaccines.</a> NPJ Vaccines. 4, 16 (2019).</li><li>Ma, Z., Zhang, H., Wang, P. G., Liu, X. W., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+adjacent+to+glycosylation+sites+impacts+immunogenicity+of+glycoconjugate+vaccine.">Peptide adjacent to glycosylation sites impacts immunogenicity of glycoconjugate vaccine.</a> Oncotarget. 9 (1), 75-82 (2018).</li><li>Grayson, E. J., Bernardes, G. J. L., Chalker, J. M., Boutureira, O., Koeppe, J. 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=A+coordinated+synthesis+and+conjugation+strategy+for+the+preparation+of+homogeneous+glycoconjugate+vaccine+candidates.">A coordinated synthesis and conjugation strategy for the preparation of homogeneous glycoconjugate vaccine candidates.</a> Angewandte Chemie (International Ed. in English). 50 (18), 4127-4132 (2011).</li><li>Pillot, A., 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=Site-Specific+Conjugation+for+Fully+Controlled+Glycoconjugate+Vaccine+Preparation.">Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation.</a> Frontiers in Chemistry. , (2019).</li><li>Neumann-Staubitz, P., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+unnatural+amino+acids+to+study+and+engineer+protein+function.">The use of unnatural amino acids to study and engineer protein function.</a> Current Opinion in Structural Biology. 38, 119-128 (2016).</li><li>Dumas, A., Lercher, L., Spicer, C. D., 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=Designing+logical+codon+reassignment+-+Expanding+the+chemistry+in+biology.">Designing logical codon reassignment - Expanding the chemistry in biology.</a> Chemical Science. 6 (1), 50-69 (2015).</li><li>Chen, H., Venkat, S., McGuire, P., Gan, Q., Fan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Development+of+Genetic+Code+Expansion+for+Posttranslational+Modification+Studies.">Recent Development of Genetic Code Expansion for Posttranslational Modification Studies.</a> Molecules (Basel, Switzerland). 23 (7), (2018).</li><li>Adumeau, P., Sharma, S. K., Brent, C., Zeglis, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specifically+Labeled+Immunoconjugates+for+Molecular+Imaging--Part+2:+Peptide+Tags+and+Unnatural+Amino+Acids.">Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.</a> Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging. 18 (2), 153-165 (2016).</li><li>Kularatne, S. A., 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=A+CXCR4-targeted+site-specific+antibody-drug+conjugate.">A CXCR4-targeted site-specific antibody-drug conjugate.</a> Angewandte Chemie (International Ed. in English). 53 (44), 11863-11867 (2014).</li><li>. Patent US20180333484 Polypeptide-Antigen Conjugates with Non-Natural Amino Acids Available from: <a target="_blank" href="https://patentscope.wipo.int/search/en/detail.jsf?docId=US233548973&amp;recNum=65&amp;docAn=15859251&amp;queryString=(GBS)">https://patentscope.wipo.int/search/en/detail.jsf?docId=US233548973&amp;recNum=65&amp;docAn=15859251&amp;queryString=(GBS)</a> (2018)</li><li>Quast, R. B., Mrusek, D., Hoffmeister, C., Sonnabend, A., Kubick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+incorporation+of+non-standard+amino+acids+using+cell-free+protein+synthesis.">Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.</a> FEBS letters. 589 (15), 1703-1712 (2015).</li><li>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=Engineering+the+Genetic+Code+in+Cells+and+Animals:+Biological+Considerations+and+Impacts.">Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts.</a> Accounts of Chemical Research. 50 (11), 2767-2775 (2017).</li><li>Brabham, R., Fascione, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysine+Amber+Stop-Codon+Suppression:+Development+and+Applications.">Pyrrolysine Amber Stop-Codon Suppression: Development and Applications.</a> Chembiochem: A European Journal of Chemical Biology. 18 (20), 1973-1983 (2017).</li><li>. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Genetic+Encoding+of+a+Non-Canonical+Amino+Acid+for+the+Generation+of+Antibody-Drug+Conjugates+Through+a+Fast+Bioorthogonal+Reaction">https://www.ncbi.nlm.nih.gov/pubmed/?term=Genetic+Encoding+of+a+Non-Canonical+Amino+Acid+for+the+Generation+of+Antibody-Drug+Conjugates+Through+a+Fast+Bioorthogonal+Reaction</a> (2019)</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Presolski, S. I., Hong, V. P., Finn, M. 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>

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 ...

The overall goal of this procedure is to produce a homogeneous glycoconjugate vaccine by combining non-canonical amino acid incorporation and click-chemistry. Genetic code expansion is a powerful tool to incorporate unnatural amino acids into proteins to modify their characteristics, to study or create new protein functions, or to have an access to protein conjugates. A codon suppression method has emerged as the most popular method to incorporate unnatural amino acids at different positions.This methodology will be here applied to the production of a protein carrier containing an unnatural amino acid 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 propargyl-lysine, PrK, is synthesized in two steps from commercial Boc-lysine.In the first step, the unprotected amino group of the Boc-lysine is convert to propargyl chloroformate. In a second step, the alpha amino group is de-protected. To synthesize the N(alpha)Boc-propargyl-lysine, first dissolve 500 milligrams of Boc-lysine in the mixture of five milliliters of aqueous one molar NaOH and five milliliters of THF in a flask and fit the flask with a silicon septum.Cool the flask in an ice bath and wait for the powder to be dissolved. Then add the propargyl chloroformate drop-wise over a two to three minutes period under stirring. Warm the reaction mixture at room temperature and continue the stirring for 10 hours.Cool down solutions of diethyl ether, aqueous one molar hydrochloric acid, and ethyl acetate. Cool down the crude reaction mixture in an ice bath. Pour the mixture in a separating funnel.Extract a mixture with fifty milliliters of diethyl ether. Extraction may result in a buildup of pressure, make sure you release any pressure buildup frequently. Collect the aqueous phase and discard the organic layer.Add cautiously, fifty milliliters of one molar hydrochloric acid to the aqueous phase in the separating funnel. Then, extract the aqueous layer twice using thirty milliliters of ethyl acetate. Collect the organic phase, Verify the presence of your compound by TLC.At this stage, it should be in the organic phase. Dry the combined organic layers over magnesium sulfate. Filter off the solid phase.And concentrate the filtrate under reduced pressure on a rotary evaporator. Dissolve sample of the crude oily N(alpha)Boc-propargyl-lysine in deuterated chloroform and control its identity by NMR. To synthesize the propargyl-L-lysine, introduce the N(alpha)Boc-propargyl-lysine in a round button flask equipped with a septum.Add four milliliters of anhydrous dichloromethane into the flask under argon. Add drop-wise, four milliliters of trifluoroacetic acid under stirring. Replace the silicon septum for a glass lid to avoid TFA from damaging the silicon septum.Stir the reaction mixture for one hour at room temperature. Verify the de-protection of the Boc-PrK by TLC. Concentrate the reaction mixture under reduced pressure.Add diethyl ether to the crude residue. Filter the propargyl-L-lysine in the form of a white solid on a fritted-glass. Dissolve an aliquot of the propargyl-L-lysine in deuterium oxide, then carry out NMR analysis to control its identity and purity.Propargyl-L-lysine is then dissolved in distilled water at the final concentration of one hundred millimolar and stored at minus 20 degrees in one milliliter aliquots. To co-transform plasmids into the expression strain, thaw a one hundred microliters aliquot of chemically competent E.Coli BL21 on ice during five minutes. Add one microliter of each plasmid or fifty to one hundred nanograms of each into the cells and incubate them for 30 minutes on ice.Incubate the competent cells at 42 degrees, during 45 seconds. Then, put them back on ice for two minutes. Add nine hundred microliters of LB medium and incubate under shaking for one hour at 37 degrees to allow antibiotics expression.Then plate the transformed bacteria on LB agar with antibiotics. Allow bacteria to grow overnight at 37 degrees. To express proteins modified with PrK, inoculate a single co-transformed colony in five milliliters of LB medium with antibiotics.Incubate overnight at 37 degrees with shaking. The next day, dilute with the five milliliters of primary culture in five hundred milliliters of auto-induction medium containing antibiotics, 0.02%of L-arabinose, and one millimolar of PrK and incubate at 37 degrees for 24 hours under shaking. Include a negative control by performing the culture without PrK and the positive control by performing the culture of a clone containing the wild-type protein gene.Harvest cells from the overnight culture by centrifugation at 5, 000 x g during 10 minutes. Discard the supernatant and freeze the pellet at minus 20 degrees. For the purification of the protein by gravity flow-bench affinity chromatography, resuspend the cell pellets into twenty milliliters of lysis buffer.Add DNase I and lysozyme, and allow lysis by incubating the suspension at 37 degrees during 30 minutes. Sonicate the cells in ice during five minutes, then remove the cell debris by centrifugation at 20, 000 x g during 30 minutes. Filtrate the lysate with 0.45 micrometer filter Add Ni-NTA resin to the suspension.Mixed gently at four degrees for one hour. Pour the suspension into a polypropylene column and collect the unbound fraction. Wash the resin with ten milliliters and then five milliliters of washing buffer.Collect the wash fractions. Elute the His-tagged protein with one millimeter of elution buffer and repeat this step four times and collect all the additional fractions. Combine the fractions containing pure histidine-tagged proteins and dialyze it into one liter of TEV protease buffer overnight by using dialysis membrane.Collect the protein sample into a 50 milliliter tube and add TEV buffer to obtain a final concentration of two milligrams per milliliter in a final volume of one milliliter. Add one hundred microliters of TEV protease. Add 50 microliters of 0.1 molar DTT.Complete with TEV buffer up to five milliliters. Incubate overnight at four degrees, with slow shaking. To remove EDTA, dialyze the protein at four degrees overnight by using dialysis membrane and phosphate buffer with five millimolar imidazole.To eliminate TEV protease and undigested proteins, incubate the mix with Ni-NTA beads and mix gently for one hour at four degrees. Pour the suspension into a polypropylene column. Collected the unbound fraction.The TEV protease and undigested proteins stay bound to the beads and the digested protein is eluted. Wash the column with five milliliters of washing buffer and collect the wash fractions as well. Dialyze the digested protein against one liter of click buffer at four degrees overnight with dialysis membrane to remove imidazole as well as to exchange the buffer.And measure the concentration of the protein at 280 nanometers. To conjugate the mPsaA with 6-hexachloro-fluorescein-azide, or an antidote-functionalized carbohydrate antigen, put the PrK mutated protein at a concentration of 57.8 micromolar into a two milliliter micro tube. Add 10 microliters of five millimolar azide compound, then add a pre-mix of copper sulfate solution and THPTA.Add aminoguanidine hydrochloride. Add extemporaneously prepared aqueous solution of sodium ascorbate. Close the tube, mix by inverting several times and incubate at room temperature for two hours.Stop the reaction by adding EDTA. Check the conjugation on SDS page. After migration, visualize the gel on UV light at 312 nanometers for the conjugate with fluorescein.Or stain the gel with Coomassie Blue to visualize the conjugate with the carbohydrate antigen. As addition of the hapten should induce a change of molecular weight. Purify the glycoconjugate by applying it to a gel-filtration column equilibrated with PBS.Collect the fractions containing the glycoconjugates. For prolonged storage, dialyze the glycoconjugate against the distilled water, then freeze-dry and store the glycoconjugate at minus 80 degrees. The PrK has been introduced at position 32 in replacement of a lysine near the N-terminus of the PsaA.The efficacy of the mPsaA production has been checked by SDS page and Western blot analysis using anti-Histidine tag antibody. The presence of a full length protein strongly indicates the successful incorporation of the PrK. The intensity is, however, lower than that observed for wild-type mPsaA.The mPsaA(K32PrK)has been purified on Ni-NTA beads. With a typical yield of eight milligrams and the incorporation of the PrK residue was finally confirmed by mass spectrometry. The Histidine tag was removed upon proteolytic cleavage using the TEV protease.Having the mPsaA(K32PrK)the reactivity of the alkyne was assessed using a azido-functionalized fluorescein and further used to conjugate a synthetic oligosaccharide antigen. Experiments were done in comparison with wild-type mPsaA as a control. Finally, the glycoconjugate was purified by gel filtration and its identity confirmed by mass spectrometry.In this project, homogeneous glycoconjugate vaccines have been prepared using the amber stop codon suppression technology to incorporate an unnatural amino acid under defined sites. Glycoconjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization. And thereby, satisfying more and more demanding drug agency recommendations.This criterion is not satisfied by using the classical pure-conjugation method. Moreover, this protocol makes it possible to finely tune the structure of the glycoconjugate vaccine. Giving rise to an unprecedented tool to study the relationship between the homogeneity and the structure of a glycoconjugate.

Research

JoVE Journal

Bioengineering

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 ...

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 ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

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. ...

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., ...

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 ...

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 ...

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 ...

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 ...