This article describes a method for biotinylating Mycobacterium bovis BCG to enhance its immune response properties. The process involves treating the bacteria with a biotinylation reagent and subsequently modifying the surface with avidin-fusion proteins.
Begin with a suspension of Mycobacterium bovis BCG, treated with a non-ionic surfactant, to prevent bacterial clumping.
Next, add a biotinylation reagent and incubate.
This biotinylation reagent, a complex of biotin complexed to a functional linker, facilitates binding biotin molecules to bacterial surface proteins, labeling bacteria.
Post-incubation, centrifuge the biotin-labeled bacterial suspension to remove unreacted biotinylation reagent.
Resuspend the bacteria in a buffer. Add a solution of avidin-fusion protein, a recombinant protein comprising a target antigen domain fused to monomeric avidin.
During incubation, avidin binds to biotin, facilitating bacterial surface modification with target antigens.
Centrifuge the suspension to remove unbound fusion proteins.
Resuspend the antigen-coated bacteria in a buffer containing a non-ionic surfactant. The antigen coating on the bacteria can enhance its property to trigger immune responses.
Work in a biosafety cabinet using personal protective equipment because BCG is a level II pathogen.
To biotinylate the surface of BCG cells, first, grow the cells on a shaker platform to the proper density. Collect about 1 billion cells, and wash them three times using 500 microliters of ice-cold endotoxin-free PBST. Then, spin down the cells, and suspend them in sterile endotoxin-free PBS. Next, prepare fresh 10-millimolar Sulfo-NHS SS biotin in sterile filtered water. Then, incubate bacteria in 1 milliliter of broth containing 0.5 millimolar of Sulfo-NHS SS biotin. Perform the incubation at room temperature for 30 minutes.
Next, wash the now labeled bacteria three times with 500 microliters of ice-cold PBST, to remove the unreacted reagent. Then, resuspend the pellet in 1 milliliter of PBST.
Mix 500 million biotinylated BCGs with avidin fusion protein, for a final concentration of 10 micrograms of protein per milliliter. Let this reaction go for an hour at room temperature on a shaker. Next, wash the bacteria three times with 500 microliters of ice-cold PBST.
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