Construction and characterization of M13 bacteriophages displaying functional IgG-binding domains of staphylococcal protein A.
Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG)-binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in separate phagemid vectors, to the 5' end of gene 111 of the phage M13. Upon infection by helper phage M13KO7, phagemid particles encapsulating single-stranded DNA were produced. Dot immunoblot and Western blot analyses showed the presence of fusion proteins on the M13 surface. Binding of rabbit IgG-horseradish peroxidase (IgG-HRP) complex to the phage particles confirmed that the fusion proteins possessed functional IgG-binding domains. The interaction of these phages with immobilised human IgG and its various subclasses was studied by the phage capture immunoassay where the captured phages were detected by a monoclonal antibody to the major coat protein encoded by gene VIII (gVIII). The phages showed maximal binding to IgG1 kappa, followed by IgG2 kappa, and showed negligible binding to the IgG3 kapa and IgG3 lambda subclasses. The specificity of IgG-binding phages was confirmed in a phage capture and elution assay where the binding of these phages to immobilised human IgG1 kapa weas abolished in the presence of excess of soluble protein A. Moreover, IgG-binding phages could be enriched approx. 1000-fold over non-specific phages in a single round of panning.(ABSTRACT TRUNCATED AT 250 WORDS)
Kushwaha
A
A
Department of Biochemistry, University of Delhi South Campus, New Delhi India.
Chowdhury
P S
PS
Arora
K
K
Abrol
S
S
Chaudhary
V K
VK
D000818:Animals; D000830:Animals, Laboratory; D017104:Bacteriophage M13; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A