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David O'Connell
Publications and source records attributed to David O'Connell.
Phage versus phagemid libraries for generation of human monoclonal antibodies.
Non-immune (naïve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naïve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.
Amino Acid Sequence↗
Three new members of the serine-aspartate repeat protein multigene family of Staphylococcus aureus.
Three new genes encoding the serine-aspartate (SD) repeat-containing proteins SdrC, SdrD and SdrE were found in Staphylococcus aureus strain Newman. The SD repeats had earlier been found in the S. aureus fibrinogen-binding clumping factors ClfA and ClfB. The clfA and clfB genes encode high-molecular-mass fibrinogen-binding proteins that are anchored to the cell surface of S. aureus. The sdr genes now reported are closely linked and tandemly arrayed. The putative Sdr proteins have both organizational and sequence similarity to ClfA and ClfB. At the N-terminus, putative secretory signal sequences precede approximately 500 residue A regions. The A regions of the Sdr and Clf proteins exhibit only 20-30% residue identity when aligned with any other member of the family. The only conserved sequence is the consensus motif TYTFTDYVD. The Sdr proteins differ from ClfA and ClfB by having two to five additional 110-113 residue repeated sequences (B-motifs) located between region A and the R-region. Each B-motif contains a consensus Ca2+-binding EF-hand loop normally found in eukaryotic proteins. The structural integrity of recombinant SdrD(B1-B5) protein comprising the five B-repeats of SdrD was shown by bisANS fluorescence analysis to be Ca2+-dependent, suggesting that the EF-hands are functional. When Ca2+ was removed the structure collapsed to an unfolded conformation. The original structure was restored by addition of Ca2+. The C-terminal R-domains of the Sdr proteins contain 132-170 SD residues. These are followed by conserved wall-anchoring regions characteristic of many surface proteins of Gram-positive bacteria. The sdr locus was present in all 31 S. aureus strains from human and bovine sources tested by Southern hybridization, although in a few strains it contained two rather than three genes.
Amino Acid Sequence↗