Rapid Fabrication of Protein Microarrays via Autogeneration and on-Chip Purification of Biotinylated Probes.

Abstract

A streamlined approach toward the rapid fabrication of streptavidin-biotin-based protein microarrays was investigated. First, using our engineered versatile plasmid (pBADcM-tBirA) and an optimal coexpression strategy for biotin ligase and biotin acceptor peptide (BAP) chimeric recombinant protein, an autogeneration system for biotinylated probes was developed. This system permitted an advantageous biotinylation of BAP chimeric recombinant proteins, providing a strategy for the high-throughput synthesis of biotinylated probes. Then, to bypass the conventional rate-limiting steps, we employed an on-chip purification process to immobilize the biotinylated probes with high-throughput recombinant lysates. The integration of the autogeneration of probes and on-chip purification not only contributed to the effective and reliable fabrication of the protein microarray, but also enabled simplification of the process and an automated throughput format. This labor- and cost-effective approach may facilitate the use of protein microarrays for diagnosis, pharmacology, proteomics, and other laboratory initiatives.