Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.
BACKGROUND
The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS).


OBJECTIVE
pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS.


RESULTS
Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR.


CONCLUSIONS
Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.
Fuchs
P
P
Recombinant Antibody Research Group, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Weichel
W
W
Dübel
S
S
Breitling
F
F
Little
M
M
D000595:Amino Acid Sequence; D002469:Cell Separation; D002473:Cell Wall / Q000737:chemistry; D004926:Escherichia coli / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D005818:Genetic Engineering; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology; D011993:Recombinant Fusion Proteins
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv