Comparison of the structural stability and dynamic properties of recombinant anthrax protective antigen and its 2-fluorohistidine-labeled analogue.
Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen's conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7-8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate.
Macromolecule and Vaccine Stabilization Center, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
Lei
L
Hu
Sangeeta B
SB
Joshi
Kiran K
KK
Andra
Santosh V
SV
Thakkar
David B
DB
Volkin
James G
JG
Bann
C Russell
CR
Middaugh
D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D002942:Circular Dichroism; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D006639:Histidine; C007749:2-fluorohistidine