Cloning of anti-Gal Fabs from combinatorial phage display libraries: structural analysis and comparison of Fab expression in pComb3H and pComb8 phage.
Anti-Gal is the most abundant natural antibody in humans. It interacts specifically with the carbohydrate epitope Gal alpha1-3Galbeta1-4GlcNAc-R (termed the alpha-galactosyl epitope). In an attempt to characterize the Ig genes encoding anti-Gal, two combinatorial phage display libraries in phagemid pComb3H were screened for anti-Gal Fabs. For this purpose, phages were incubated with biotinylated BSA coupled with alpha-galactosyl epitopes (designated alpha-Gal-BSA). Subsequently, phages complexed with alpha-Gal-BSA were isolated by streptavidin-coupled magnetic beads. Because of the low affinity of this antibody, a characteristic shared with other anti-carbohydrate antibodies, only two clones displaying anti-Gal activity were isolated. Clone G9 contained the VH gene V3-43 and VL gene DPK15, whereas clone P19 contained the VH gene V3-15 and VL gene DPL16. Both clones contained between five and 14 mutations in their H and L chain V genes. The affinity of clone G9 was found to be higher than that of clone P19, as only the former could bind to solid-phase alpha-galactosyl epitopes in an enzyme-linked immunosorbent assay. This interaction could be increased by expressing Fabs in phagemid pComb8 grown in the presence of IPTG. Under such conditions, the IPTG-activated Lac-Z promoter induces an increased expression of Fabs that are linked to phage envelope protein VIII, resulting in multiple Fab display on the phage. The data suggest that screening combinatorial phage display libraries for anti-carbohydrate antibodies may be more effective with pComb8 phage grown in the presence of IPTG.
Wang
L
L
Department of Microbiology and Immunology, Allegheny University, Philadelphia, PA 19129, U.S.A.
Radic
M Z
MZ
Siegel
D
D
Chang
T
T
Bracy
J
J
Galili
U
U
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014312:Trisaccharides / Q000276:immunology*
D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D007544:Isopropyl Thiogalactoside