High‐throughput analysis of GST‐fusion protein expression and activity‐dependent protein interactions on GST‐fusion protein arrays with a spectral surface plasmon resonance biosensor

Abstract

We modified gold arrays with a glutathione (GSH) surface, and investigated high-throughput protein interactions with a spectral surface plasmon resonance (SPR) biosensor. We fabricated the GSH exterior on gold surfaces by successive modification with aminoethanethiol, 4-maleimidobutyric acid N-hydroxysuccinimide ester and GSH. We immobilized GST-Rac1, GST-RhoA, the GST-Rho-binding domain of rhotekin and the GST-p21-binding domain of PAK1 onto the GSH surface, and observed specific antigen-antibody interactions on the GST-fusion protein arrays. We determined the expression of GST-fusion proteins in Escherichia coli on the GSH surface with the SPR biosensor. We then analyzed the interactions of tissue transglutaminase (tTGase), a Ca2+-dependent enzyme, with RhoA and Rac1 on the GST-fusion protein arrays with the SPR biosensor. We found that tTGase interacted with RhoA and Rac1 in a Ca2+-dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca2+ concentration. We obtained similar results with GST pull-down assays. Thus, protein arrays prepared on the GSH surface provide a useful system for the high-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions with the spectral SPR biosensors.

Protocols of proteins interactomics: molecular fishing on optical chips and magnetic nanoparticles

“…2А). Использование GST-метки обеспечивает высоко специфическое и прочное связывание меченого белка на сорбенте с глутатионом [14][15][16][17]. Это обусловлено специфичностью и прочностью связывания субстрата (глутатион) в активном центре фермента (GST).…”

Surface‐Plasmon‐Resonance‐Based Chemical Proteomics: Efficient Specific Extraction and Semiquantitative Identification of Cyclic Nucleotide‐Binding Proteins from Cellular Lysates by Using a Combination of Surface Plasmon Resonance, Sequential Elution and Liquid Chromatography–Tandem Mass Spectrometry

“…When SPR is combined with MS, quantitative and qualitative information can be combined, and a powerful technique is obtained that is suitable for the determination of intact proteins [11][12][13][14] as well as digests. [15][16][17][18] Although beads and SPR generally result in comparable extraction, [7,9,17] it has been hypothesized that the nonspecific binding on SPR discs is reduced due to the hydrophilic and nonporous characteristics of the sensor, and the high level of functional groups attached. [17] Similar to other affinity purifications, nonspecific binding is a major challenge when more complex protein sources like cell lysate or plasma are presented to the SPR sensor.…”

“…SPR can be used as an affinity-based extraction technique in which the extraction process can be followed in real time, without the need to label proteins. [7][8][9][10] More importantly, SPR provides quantitative information about the amount of material bound to the sensor. When SPR is combined with MS, quantitative and qualitative information can be combined, and a powerful technique is obtained that is suitable for the determination of intact proteins [11][12][13][14] as well as digests.…”

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

“…immunosensor. BSA is added to immunosensors to block free Protein G molecules to reduce non-selective binding (Dong et al, 2008;Jung et al, 2006;Jung et al, 2007;Lu et al, 2001). The addition of BSA increases the thickness of the layers which can decrease the sensitivity of the SPR response.…”

“…Therefore, the sensor without BSA was more sensitive. BSA has been shown to decrease the sensitivity of other immunosensors (Dong et al, 2008;Jung et al, 2006;Jung et al, 2007;Lu et al, 2001).…”