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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Synthesis of the UAA: propargyl-lysine (PrK)

1. Synthesis of N^α-Boc-propargyl-lysine¹⁸
   1. 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.
   2. Cool the flask in an ice bath and then add 158 µL of propargyl chloroformate (1.62 mmol) dropwise (over a 2-3 min period) using a microsyringe while stirring.
   3. Warm the reaction mixture to .......

Discussion

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 vaccines^7,8,14. 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.......

Materials

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