tailieunhanh - Báo cáo Y học: Human immunoglobulin A (IgA)-specific ligands from combinatorial engineering of protein A

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid, three-helix bundle domain derived from the IgG-binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. . | Eur. J. Biochem. 269 2647-2655 2002 FEBS 2002 doi Human immunoglobulin A IgA -specific ligands from combinatorial engineering of protein A Jenny Ronnmark1 Hans Gronlund2 Mathias Uhlen1 and Per-Ake Nygren1 1 Department of Biotechnology Royal Institute of Technology SCFAB Stockholm Sweden 2Department of Medicine Unit of Clinical Immunology and Allergy Karolinska nnsittute Sweden Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins affibodies shwifing selcctive bidding to umnan IgA. From protein libraries constructed by combinatorial mutagenesis of a 58-amino-acid three-helix bundle domain derived from the IgG-binding staphylococcal protein A variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies myeloma as tareet looli culf s. S aa LlC tel inLitian of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding with dissociation constants Kd in the range between and 3 M. One variant ZIgA1 soo hire sterneest binding affinity was further analyzed and showed that human IgA subclasses IgA1 and IgA2 as well as secretory IgA were recognized with similar efficiencies. No detectable crossreactivity towards human IgG IgM IgD or IgE was observed. The potential use of the ZIgA1 affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate using an affinity column containing a divalent head-to-tail ZIgA1 affibody dimer construct as a ligand. In addition efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated. Keywords IgA affibody combinatorial protein engineering affinity ligand phage display. Efficient