Tyrosine‐based “Activatable Pro‐Tag”: Enzyme‐catalyzed protein capture and release

Abstract

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.

Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro‐tag

“…This agrees with previously reported results (Chen et al, 2003a;Lewandowski et al, 2006) which examined GFP binding to chitosan in solution. Electrodeposited chitosan may have structural and chemical properties that are distinctly different than soluble chitosan, particularly in that the chitosan experiences a high pH for a short time during the electrodeposition.…”

“…Finally, we expect the vast majority of GFP molecules to be specifically oriented through the C-terminal pro-tags as they assemble onto the scaffold surface, as we previously demonstrated that GFP preferentially grafts to chitosan through the pro-tag versus native tyrosines by a ratio of over 4:1; that is, over 80% of binding is through the pro-tag (Lewandowski et al, 2006). This enables orientational control and contrasts considerably with an additional previous approach, where the protein was covalently linked through its native amines to a glutaraldehyde-activated chitosan film surface and thus in no particular orientation (Yi et al, 2005a).…”

“…The chip was then incubated in both tyrosinase (0.1 mg/mL or 166 Units/mL) and (His) 6 -GFP-EK-(Tyr) 5 (0.2 mg/mL) in PBS for 16 h at 48C. The production of (His) 6 -GFP-EK-(Tyr) 5 was previously described (Lewandowski et al, 2006). Briefly, the GFP was expressed in E. coli BL21, and IMAC-purified using a 5 mL HiTrap Chelating HP column charged with Ni 2þ ions (GE Healthcare BioSciences, Uppsala, Sweden).…”

“…The target model protein is green fluorescent protein (GFP). GFP preferentially grafts to the chitosan scaffold through its C-terminal pro-tag versus its native tyrosines by a ratio of over 4:1 (Lewandowski et al, 2006); that is, over 80% of GFP molecules are grafted through the C-terminal pro-tag. Thus the GFP molecules are specifically oriented through the pro-tag with respect to the scaffold surface and are subsequently accessible in aqueous solution.…”

Towards area‐based in vitro metabolic engineering: Assembly of Pfs enzyme onto patterned microfabricated chips

“…In the second method, the Pfs predominately conjugates to the surface of the electrodeposited chitosan film, as Pfs is presented after chitosan deposition, and as diffusion of Pfs into the film is presumably limited due to steric hindrance effects. Additionally, Pfs preferentially conjugates to chitosan film through its C-terminal protag vs. its native tyrosines, 34 indicating that Pfs is preferentially oriented through the pro-tag with respect to the film surface. Thus, the Pfs assembled by the second method is expected to be more accessible for antibody binding, which is consistent with the strong fluorescence intensity of Figure 2b.…”