Screening and Characterization of Affinity Peptide Tags Specific to Polystyrene Supports for the Orientated Immobilization of Proteins

Abstract

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.

Efficient immobilization of a ligand antibody with high antigen-binding activity by use of a polystyrene-binding peptide and an intelligent microtiter plate

“…In previous studies, we screened and characterized peptides that interact specifically with the surface of hydrophilic PS plates (Kumada et al, 2006(Kumada et al, , 2007a of PS-tags to target proteins by genetic fusion or chemical conjugation allowed site-specific immobilization of the proteins directly onto the hydrophilic PS plates, while retaining high biological activity, especially when non-ionic surfactants, such as Tween 20 and Triton X-100, were present during the adsorption step (Kumada et al, in press). Furthermore, one-step and two-step detection methods that utilized specific recognition of the introduced PS-tags for the hydrophilic PS surface reduced assay time without loss of sensitivity (Kumada et al, 2007a,b).…”

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

“…Although, there are many methods for protein immobilization on to solid surfaces, site-specific protein immobilization has been increasingly used to maintain protein conformation and retained biological activity on selected solid surfaces (Kumada et al, 2006;Kwon et al, 2006;Park et al, 2006). Existing protein immobilization methods are mostly based on non-specific adsorption via, for example, intermolecular forces, mainly ionic bonds and hydrophobic and polar interactions, or chemical coupling reactions which mostly lead to uncontrolled attachment of the proteins on to solid surfaces (Kwon et al, 2006;Rusmini et al, 2007).…”

“…Recently they are also used as part of a fusion protein in the development of multifunctional molecular constructs for a variety of practical applications (Dai et al, 2005;Holmes, 2002;Johnson et al, 2008;Kramer et al, 2004;Sanchez et al, 2005). Such hybrid platforms, containing genetically fused proteins with site-specific solid-binding peptides, could be potential novel ways of efficient immobilization of enzymes and receptors under ambient conditions with wide variety of applications (Brown, 1997;Ishikawa et al, 2008;Krauland et al, 2007;Kumada et al, 2006;Park et al, 2006;Zhang and Cass, 2001).…”

Poly(vinylpyrrolidone-b-styrene) block copolymers tethered surfaces for protein adsorption and cell adhesion regulation

“…Indeed, the use of surface PVP has come to be recognized as effective in resisting nonspecific protein adsorption [18]. Polystyrene has been extensively utilized as a common substrate for protein immobilization [19]. Moreover, since there are hydrophilic and hydrophobic domains on the surfaces of proteins, the significant wettability differences between the two blocks may influence ensuing interactions with proteins.…”