Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.
Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry. In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed. Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity. Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems. In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number. Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity. This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics. Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E. coli surface displayed libraries.
Daugherty
P S
PS
Department of Chemical Engineering, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Olsen
M J
MJ
Iverson
B L
BL
Georgiou
G
G
D000906:Antibodies / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology; D003598:Cytoskeletal Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D003598:Cytoskeletal Proteins; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011506:Proteins; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins; D001616:beta-Galactosidase