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.

Site-specific Effector Protein Functionalization to Create Bead-based Avidity Model Systems

“…Fusion of MBP to BirA resulted in reproducible in vivo biotinylation of heterologously expressed proteins in E. coli (49). Previous reports indicated a reduction in the growth rate of E. coli when BirA is overexpressed (50), however under conditions described here, no significant reduction in the growth rate was observed (Supplemental Figure S1). Using our previously described purification conditions (51) resulted in a stable complex of MBP-BirA and GFP-CBMs during IMAC purification (Supplemental Figure S2, lane 2), most likely due to the presence of salts (500 mM) and absence of polymers which are known to reduce protein-protein interactions.…”

“…This represents a significant advantage over in vitro biotinylation, which requires one buffer exchange step into suitable buffer for biotinylation and a second purification/ buffer exchange step to remove excess biotin and BirA (65). This convenient in vivo biotinylation method could be applied to express a library of protein variants in microplate-based cell cultures (50), with the potential to automate the purification process using suitable liquid handling systems (66). The purified protein variants could then directly be combined with streptavidin-coated beads for screening or characterization using a highly multiplexed AFS or other bead-based assays.…”