Bacteriophage surface display of an immunoglobulin-binding domain of Staphylococcus aureus protein A.
As a model system for the optimization of separation ligands by bacteriophage surface display, we have constructed a phage surface expression system for a single immunoglobulin-binding domain of Protein A of Staphylococcus aureus. Protein A domain B is genetically fused to the gpIII adsorption protein of the filamentous bacteriophage M13, and hence displayed on the phage surface. Phage displaying the Protein A domain are selectively retained on human IgG-sepharose. Retention is due to specific Protein A-IgG interactions, as demonstrated by competitive inhibition by soluble Protein A or polyclonal human IgG. Polyclonal goat IgG, which is known to bind less well to Protein A than does human IgG, inhibits phage adsorption less effectively. Phage expressing Protein A can be purified in a few rounds of selective adsorption from a vast excess of wild type phage. Diverse libraries constructed by mutagenesis of this construct will allow massive screening of mutant forms of Protein A for alterations in binding and elution properties. We anticipate that phage display will prove to be a widely-applicable method of identification and optimization of affinity ligands for separations.
Djojonegoro
B M
BM
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204.
Benedik
M J
MJ
Willson
R C
RC
D000595:Amino Acid Sequence; D017104:Bacteriophage M13; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D005813:Genes, Synthetic; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A