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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 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.
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....
1. Synthesis of the UAA: propargyl-lysine (PrK)
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
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.......
E.C. gratefully acknowledges the financial support from La Région Pays de la Loire (Pari Scientifique Program "BioSynProt"), 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.
....Name | Company | Catalog Number | Comments |
AIM (autoinductif medium) | Formedium | AIMLB0210 | Solid powder |
Boc-Lys-OH | Alfa-Aesar | H63859 | Solid powder |
BL21(DE3) | Merck Novagen | 69450 | E. coli str. B, F- ompT gal dcm lon hsdSB(rB-mB-) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) |
Dialysis membrane | |||
DNAseI | |||
Filter 0.45 µm | |||
L-arabinose | |||
lysozyme | |||
Ni-NTA resin | Machery Nagel | Protino | Ni-NTA beads in suspension into 20% ethanol |
Pall centrifugal device | |||
pET24d-mPsaAK32TAG-ENLYFQ-HHHHHH | this study | same as pET24d-mPsaA-WT but with a K32TAG mutation in the mPsaA gene | |
pET24d-mPsaA-WT | this study | 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 | |
pEVOL plasmid | gift fromEdward Lemke EMBL (ref 19) | 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 | |
Propargyl chloroformate | Sigma-Aldrich | 460923 | Liquid |
Sonicator | Thermo Fisher | FB120-220 |
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