Engineering regulable Escherichia coli beta-galactosidases as biosensors for anti-HIV antibody detection in human sera.
The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.
Ferrer-Miralles
N
N
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Feliu
J X
JX
Vandevuer
S
S
Müller
A
A
Cabrera-Crespo
J
J
Ortmans
I
I
Hoffmann
F
F
Cazorla
D
D
Rinas
U
U
Prévost
M
M
Villaverde
A
A
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D020134:Catalytic Domain; D004795:Enzyme Stability; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology; D015483:HIV Antibodies / Q000097:blood*; D015700:HIV Envelope Protein gp41 / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase / Q000235:genetics
D000939:Epitopes; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase