title: The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis.
abstract: Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.
authors.affiliation: Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Samer
authors.initials: S
authors.lastname: Singh
authors.forename: Mohd Azhar
authors.initials: MA
authors.lastname: Aziz
authors.forename: Puneet
authors.initials: P
authors.lastname: Khandelwal
authors.forename: Rajiv
authors.initials: R
authors.lastname: Bhat
authors.forename: Rakesh
authors.initials: R
authors.lastname: Bhatnagar
mesh_terms: D000818:Animals; D022122:Anthrax Vaccines; D000942:Antigens, Bacterial / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D002460:Cell Line; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D008745:Methylation; D051379:Mice; D009995:Osmosis; D013696:Temperature
chemical_list: D022122:Anthrax Vaccines; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D005998:Glycine