A single Fc binding domain--alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity.
A recombinant gene fusion was created and cloned using a previously constructed gene encoding a monodomain IgG Fc binding protein and the gene coding for bacterial alkaline phosphatase. The construct was able to express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of the protein demonstrating both biological activities was detected from periplasmic space preparations. Nanogram concentrations of the Fc binding--alkaline phosphatase fusion protein allowed primary IgG antibody detection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resistance of the protein to proteolytic degradation and the loss of IgG Fc binding ability. Using affinity-purified fusion protein, the specificity of binding to IgG, IgM and IgA was examined; binding was strong to IgG and barely detectable against IgM or IgA. Affinity for binding of the fusion protein to IgG (Kd = 6.7 x 10(-8) M) was determined to be equal to or greater than previously reported for protein A.
Wang
C L
CL
Department of Bacteriology/Biochemistry, University of Idaho, Moscow 83844.
Huang
M
M
Wesson
C A
CA
Birdsell
D C
DC
Trumble
W R
WR
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D007070:Immunoglobulin A / Q000378:metabolism; D007127:Immunoglobulin Constant Regions / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D007075:Immunoglobulin M / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007070:Immunoglobulin A; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins; C089103:SCIB protein, recombinant; D000469:Alkaline Phosphatase