Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.
We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.
Christmann
A
A
Abteilung Molekulare Genetik und Präparative Molekularbiologie, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077, Göttingen, Germany.
Wentzel
A
A
Meyer
C
C
Meyers
G
G
Kolmar
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology; D006692:Classical Swine Fever Virus / Q000235:genetics / Q000276:immunology; D006224:Cricetinae; D004274:DNA, Recombinant / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D005434:Flow Cytometry; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D019151:Peptide Library; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D004274:DNA, Recombinant; D019151:Peptide Library; D014759:Viral Envelope Proteins