A surface expression vector for antibody screening.
To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili. Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG. Ab::pIII was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences. It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen. The phagemid DNA is even smaller than that of wt fd phages and can easily be propagated in plasmid form. Most importantly, its Ab::pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants. After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.
Breitling
F
F
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Dübel
S
S
Seehaus
T
T
Klewinghaus
I
I
Little
M
M
D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D036022:Capsid Proteins; D002462:Cell Membrane / Q000276:immunology; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D015698:Genomic Library; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014760:Viral Fusion Proteins; D014764:Viral Proteins / Q000235:genetics
D000906:Antibodies; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D004247:DNA