Regioselective Covalent Immobilization of Recombinant Antibody-Binding Proteins A, G, and L for Construction of Antibody Arrays

• Published in: Journal of the American Chemical Society 2013-06, Vol.135 (24), p.8973-8980
• Main Authors: Seo, Jin-soo, Lee, Sungwon, Poulter, C. Dale
• Format: Article
• Language: English
• Subjects: alkynes; antibodies; Antibodies - analysis; antigens; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; binding proteins; buffers; Cloning, Molecular; copper; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; Escherichia coli; Escherichia coli - genetics; geranylgeranyl diphosphate synthase; glass; hydrochloric acid; Immobilized Proteins - chemistry; Immobilized Proteins - genetics; microarray technology; Peptostreptococcus - chemistry; Peptostreptococcus - genetics; Protein Array Analysis - methods; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Staphylococcal Protein A - chemistry; Staphylococcal Protein A - genetics; Staphylococcus aureus - chemistry; Staphylococcus aureus - genetics; Stereoisomerism; Streptococcus - chemistry; Streptococcus - genetics; washing
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja402447g

Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies.