In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: a strategy for efficient display of large full-length proteins.
An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc(-) phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.
Shivachandra
Sathish B
SB
Department of Biology, The Catholic University of America, Washington, DC 20064, USA.
Rao
Mangala
M
Janosi
Laszlo
L
Sathaliyawala
Taheri
T
Matyas
Gary R
GR
Alving
Carl R
CR
Leppla
Stephen H
SH
Rao
Venigalla B
VB
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002213:Capsid / Q000276:immunology / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008808:Mice, Inbred CBA; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D036022:Capsid Proteins; C108525:HOC protein, T4 phage; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin