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

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.