       Document 0045
 DOCN  M95A0045
 TI    A molecular sensor system based on genetically engineered alkaline
       phosphatase.
 DT    9510
 AU    Brennan CA; Christianson K; La Fleur MA; Mandecki W; Abbott
       Laboratories, North Chicago, IL 60064-4000, USA.
 SO    Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5783-7. Unique Identifier :
       AIDSLINE MED/95320159
 AB    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.
 DE    Alkaline Phosphatase/*CHEMISTRY/ISOLATION & PURIF/*METABOLISM  Amino
       Acid Sequence  Antibodies  Antigenic Determinants/ANALYSIS  Aspartic
       Acid  Binding Sites, Antibody  Binding, Competitive
       Capsid/CHEMISTRY/PHARMACOLOGY  Comparative Study  Escherichia
       coli/ENZYMOLOGY/GENETICS  Genes, Bacterial  Genetic Engineering  Glycine
       Hepatitis C Viruses  HIV Envelope Protein gp120/CHEMISTRY/PHARMACOLOGY
       HIV-1  Molecular Sequence Data  Mutagenesis, Site-Directed  Peptide
       Fragments/PHARMACOLOGY  Point Mutation  Protein Conformation  Protein
       Hybridization  Recombinant Proteins/*CHEMISTRY/ISOLATION &
       PURIF/METABOLISM  Serine  Viral Core Proteins/CHEMISTRY/PHARMACOLOGY
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

