A molecular sensor system based on genetically engineered alkaline phosphatase.
Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.
Brennan
C A
CA
Abbott Laboratories, North Chicago, IL 60064-4000, USA.
Christianson
K
K
La Fleur
M A
MA
Mandecki
W
W
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000906:Antibodies; D001224:Aspartic Acid; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002213:Capsid / Q000737:chemistry / Q000494:pharmacology; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005998:Glycine; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000494:pharmacology; D015497:HIV-1; D016174:Hepacivirus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014758:Viral Core Proteins / Q000737:chemistry / Q000494:pharmacology
D000906:Antibodies; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D001224:Aspartic Acid; D012694:Serine; D000469:Alkaline Phosphatase; D005998:Glycine