We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, through the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjugation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.

Fusion tags are widely used in the recovery and purification of recombinant proteins. We have investigated the capture and release of two fusion proteins from cell extracts using the aminopolysaccharide chitosan. We have fused to green fluorescent protein (GFP) and to S-ribosylhomocysteinase (LuxS) a "pro-tag" consisting of five tyrosine residues that are "activated" by tyrosinase-catalyzed conversion into reactive oquinones. The o-quinones react with the amino groups of chitosan, resulting in the covalent conjugation of the fusion protein to chitosan. The fusion protein is captured from solution by precipitation of the protein-chitosan conjugate due to the decrease in solubility of chitosan at higher pH. Additionally, chitosan is used to "pre-precipitate" cell extract contaminants such as nucleic acids and phospholipids, and thus, crudely purify the fusion protein remaining in solution. Finally, we released the fusion protein from chitosan back into solution using the chitosan-hydrolyzing enzyme chitosanase as an alternative to protease cleavage.