Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding.
A gene encoding a bacterial IgG Fc binding domain was designed and synthesized. The synthetic DNA fragment was cloned 3' to an inducible trpE promoter such that expression of the gene in Escherichia coli produced abundant Fc binding protein fused to the first seven amino acids of the trpE protein. The recombinant protein contained a single Fc binding domain and demonstrated efficient binding to human IgG in Western blot analysis. This protein degraded rapidly following cell lysis in the absence of protease inhibitors, but could be effectively protected by the addition of protease inhibitor. After purification of the protein by IgG affinity chromatography, IgG Fc binding ability was retained for at least 24 h at either 23 or 37 degrees C and on heating for 15 min at temperatures up to 65 degrees C. No immunoprecipitation was observed in interactions between the monodomain Fc binding protein and IgG molecules. Unlike staphylococcal protein A, no detectable binding of the monodomain IgG Fc binding protein was observed to either IgM or IgA. Truncated proteins, expressed from a series of 3' deletions of the synthetic gene, were used to estimate the minimum portion of a monodomain Fc binding protein that retained Fc binding ability.
Trumble
W R
WR
Department of Bacteriology and Biochemistry, University of Idaho, Moscow 83844.
Huang
M
M
West
J W
JW
Reasoner
J L
JL
Huang
J L
JL
Wang
C L
CL
Douthart
R J
RJ
Birdsell
D C
DC
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; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007127:Immunoglobulin Constant Regions / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011961:Receptors, Fc / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C089103:SCIB protein, recombinant; D013205:Staphylococcal Protein A