Controlled and Oriented Immobilization of Protein by Site-Specific Incorporation of Unnatural Amino Acid

Abstract

Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis of Rab1 binding onto the DrrA-immobilized surfaces using surface plasmon resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis.

Enhanced Enzyme Stability Through Site-Directed Covalent Immobilization

“…Different from prior immobilization techniques, as demonstrated here, the PRE-CISE system is an entirely covalent immobilization that requires only a single substitution or insertion to facilitate immobilization orientation control (Ewers et al, 2005;Kalia et al, 2007;Lim et al, 2014;Seo et al, 2011;Smith et al, 2005). We have demonstrated that by controlling the immobilization orientation, greater enzyme stability can be realized than nonspecific covalent immobilization, even by 50 and 73%.…”

“…Other sitespecific immobilization technologies have enabled immobilization at many potential residues, but rely on noncovalent techniques (Kalia et al, 2007;Lim et al, 2014;Seo et al, 2011). To our knowledge, no covalent site-specific immobilization method has been previously demonstrated enabling immobilization at residues throughout the enzyme while only requiring a single substitution or insertion.…”

BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells

“…However, there is more than one reactive group in BMP2, and this method might suffer from poor site-specificity and heterogeneous products. In addition, non-specific chemical modifications can result in inactivation of proteins or their incorrect orientation 18–21 .…”

Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

“…We chose Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in this study, since it is a high-yielding and reliable reaction forming a stable triazole linkage between biologically inert azide and terminal alkyne groups [16], and readily compatible with aqueous and mild conditions beneficial for protein bioconjugation [17]–[19]. CuAAC has been embraced for numerous biomolecular conjugation applications since its discovery in 2002 [20]–[23].…”