Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis.
Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48h at 37 degrees C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains approximately 45% and Phe554Ala retains approximately 90% activity, even after incubation at 37 degrees C for 48h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life.
Centre for Biotechnology, Jawaharlal Nehru University, New Mehrauli Road, 110067, New Delhi, India.
Samer
S
Singh
Nidhi
N
Ahuja
Vibha
V
Chauhan
E
E
Rajasekaran
Syed
S
Mohsin Waheed
Rajiv
R
Bhat
Rakesh
R
Bhatnagar
D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D002460:Cell Line; D005973:Glutamine / Q000502:physiology*; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000502:physiology*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D005973:Glutamine; D010649:Phenylalanine