The isolation of U1 RNA-binding antibody fragments from autoimmune human-derived bacteriophage display libraries.
Almost seventy percent of systemic lupus erythematosus (SLE) patients produce autoantibodies specific for U1 RNA, the RNA component of the U1snRNP complex involved in pre-mRNA splicing. Human anti-U1 RNA antibodies from SLE patients provide an ideal model for studying protein-RNA interactions. In addition, a more in depth look at the mechanism by which an antibody interacts with U1 RNA will lend insight into immune disregulation and may have therapeutic potential in the treatment of autoimmune disease. The possibility of cloning an anti-U1 RNA antibody has been investigated using "phage Fab display," a method involving the display of Fab fragments on the outer surface of filamentous phage. Human Fab cDNA libraries were constructed using total RNA collected from leukophoresed anti-U1 RNA antibody-positive SLE patient sera. The resulting Fab genes were subcloned into a phagemid expression vector which produced phage displaying Fab fragments in E.coli1. Libraries were enriched for U1 RNA-binding clones after several rounds of affinity selection against purified, in vitro transcribed U1 RNA. Putative U1 RNA-binding clones were identified by colony lift and the corresponding Fab genes were expressed and purified from E.coli for binding studies. Results demonstrate that RNA-binding Fab can be isolated from combinatorial phage display libraries.
Powers
J E
JE
Department of Biochemistry, University of Missouri School of Medicine, Columbia 65212, USA.
Marchbank
M T
MT
Deutscher
S L
SL
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification; D008180:Lupus Erythematosus, Systemic / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D012342:RNA, Small Nuclear / Q000276:immunology*
D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D012342:RNA, Small Nuclear