Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. crucial for development of immunobiosensors that can detect a wide variety of analytes including drugs, toxins, bacteria, pathogens, and metabolic biomarkers.17-19 Among the many strategies that have been reported is the use of immobilized forms of antibody binding proteins from (protein A), (protein G), and (protein L) to bind the antibodies to the surface.20 Proteins A and G bind to the Fc region in the heavy chains, while protein L binds to -light chains outside of the antigen binding site. Structural studies show that well-defined motifs C domains E, D, A, B, and C CI-1033 in protein A; C1, D1, and C2 in protein G; B1, B2, B3, and B4 in protein L C are responsible for binding.21 Proteins A, G, and L bind a wide variety of antibodies with different affinities (see Table S1) and have been used to immobilize antibodies in protein-based microarrays.22, 23 The sensitivity of antibody-based assays is increased when the antibodies are immobilized in well-controlled orientations that expose the antigen binding site with enhancements of as much as 100-fold for antibodies bound to immobilized protein A versus randomly oriented binding.24 Several strategies have been used to immobilize antibody-binding proteins, including hydrophobic interactions between a glass surface and protein A, G, or L fusion proteins, cysteine thiol-gold interactions, crosslinking through cysteine and lysine residues, and complementary DNA-DNA interactions.25-28 We now describe procedures for covalently immobilizing truncated versions of proteins A, G, and L, which retain their respective antibody binding motifs, in a well-defined orientation using a regiospecific chemoenzymatic approach based on bioorthogonal reactions that eliminate the need to purify the recombinant proteins at any stage of the process. Glass slides coated with the recombinant proteins A, G, and L selectively bind their respective antibody targets and can be stripped of the antibodies and reused. EXPERIMENTAL SECTION Construction of expression plasmids for antibody-binding proteins with C-terminal CVIA tags Locations encoding the antibody-binding sites in proteins A, G, and L in plasmids SpA-pGEX-KG, proLG-pHD389, and proL-pHD389,29-31 respectively, had been subcloned into pET-28b(+) (Novagen) and each was improved to append a C-terminal RTRCVIA farnesyl transferase identification site towards the portrayed proteins. A 885 bp area coding the E, D, A, B, and C domains of proteins A was amplified with PfuUltra HF DNA polymerase (Stratagene) using primers filled with a 5 NdeI site and a 3 XhoI site (find SI). PCR circumstances (30 CPP32 cycles) had been: preliminary denaturation 95 C, 120 s; denaturation, 95 C, 30 s; annealing 52.1 C, 60 s; expansion 72 C, 60 s; last expansion 72 C, 10 min. The PCR items had been purified on 1% agarose and extracted using the GFX PCR DNA gel purification package (GE health care). The purified PCR fragments and pET28b(+) had been doubly digested with NdeI and XhoI and ligated using T4 DNA ligase (New Britain Biolabs) CI-1033 to provide plasmid pProA-CVIA. XL10-Silver (Stratagene) was changed with pProA-CVIA and specific colonies were selected from LB/Kan (LB with 30 g/mL kanamycin) plates. Plasmid in the transformants was sequenced and isolated to verify the structure from the build. The encoded proteins (proA-CVIA) included five antibody-binding domains CI-1033 (E, D, A, B, and C) from proteins A using a His6-label and a thrombin proteolytic site on the N-terminus and a RTRCVIA farnesyl identification site on the C-terminus. A four amino acidity EEDN series was inserted between RTRCVIA and C-domain. PCR had not been successful with no EEDN insert because of the high homology between domains E, A, D, and B and domains C. Similar appearance plasmids for CVIA-tagged proteins G (proG-CVIA) and proteins L (proL-CVIA) had been built using same method defined for proA-CVIA with minimal adjustments. A 375 bp series encoding the C1, D1, and C2 domains of C40 proteins G was amplified with PfuUltra HF DNA polymerase (Stratagene) using primers filled with a 5 NdeI site and a 3 XhoI site (find SI) and ProLG-pHD389 being a PCR design template. The B1, B2, B3, and B4 domains of proteins L had been amplified with PfuUltra HF DNA polymerase (Stratagene) using primers filled with a 5 NdeI site and a 3 XhoI site (find SI) and ProLG-pHD389 being a PCR template. The PCR circumstances were similar with those employed for pProA-CVIA aside CI-1033 from the annealing methods; 43.7 C for the protein G plasmid and 47.1.