Protein Nanopatterns and Biosensors Using Gold Binding Polypeptide as a Fusion Partner

Abstract

An efficient strategy for immobilizing proteins on a gold surface was developed by employing the gold binding polypeptide (GBP) as a fusion partner. Using the enhanced green fluorescent protein (EGFP), severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVme), and core streptavidin (cSA) of Streptomyces avidinii as model proteins, specific immobilization of the GBP-fusion proteins onto the gold nanoparticles and generation of protein nanopatterns on the bare gold surface were demonstrated. The GBP-fused SCVme bound to gold nanoparticles successfully interacted with its antibody and showed changes in absorbance and color, allowing efficient diagnosis of SARS-CoV. The fusion proteins could be successfully immobilized on the gold surface by nanopatterning and microcontact printing as examined by atomic force microscopy and surface plasmon resonance analysis. The poly(dimethylsiloxane) microfluidic channels were created on the gold surface and were used for antigen-antibody and DNA-DNA interaction studies. Specific immobilization of GBP-EGFP fusion protein and its interaction with the antibody in the microchannels could be demonstrated. By immobilizing the DNA probe through the use of GBP-fused cSA, specific hybridization of the target DNA prepared from Salmonella could also be achieved. The GBP-fusion method allows immobilization of proteins onto the gold surface without surface modification and in bioactive forms suitable for studying protein-protein, DNA-DNA, and other biomolecular interaction studies. Furthermore, these studies can be carried out in a microfluidic system, which allows high-throughput analysis of biomolecular interactions.

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

“…By engineering this peptide in triple-repeated form (3rGBP 1 ), its binding affinity was further enhanced. The binding and assembly characteristics of 3rGBP 1 have been studied in detail by our and other groups [21,23,[31][32][33]. Here, time-dependent in situ adsorption experiments of 3rGBP 1 were carried out by applying an external electrical field using electrochemical dissipative quartz crystal microbalance (EQCM-Z) system [34,35].…”

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).…”

“…Moreover, this approach requires complex chemistries for efficient coupling (Norde, 1986;Zhen et al, 2006) which, often, may not be biocompatible. There are also further limitations in these process in which, for example, in the case of gold surface, alkanethiol layers rapidly degrade due to oxidation (Park et al, 2006;Willey et al, 2005). Current approaches, therefore, are a result of a compromise between maintaining high activity of the enzyme while having the advantage of solid immobilization via self-assembly (Bornscheuer, 2003).…”

“…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).…”

“…Recently, immobilization of enhanced green fluorescent protein, severe acute respiratory syndrome (SARS) coronavirus envelope protein and core streptavidin of Streptomyces avidinii on gold surface was achieved in conjunction with GBP1-6Histidine as fusion partner (Park et al, 2006). However, histidine-tags are known to bind to gold substrate (Peelle et al, 2005;Presnova et al, 2000;Slocik and Wright, 2003).…”