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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.
Proteomics. 2006 Feb; 6(4):1110-20.P

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.

Authors+Show Affiliations

Jung JW
Department of Molecular and Cellular Biochemistry, Kangwon National University College of Medicine, Chuncheon, Kangwon-Do, Korea.
Jung SH
No affiliation info available
Kim HS
No affiliation info available
Yuk JS
No affiliation info available
Park JB
No affiliation info available
Kim YM
No affiliation info available
Han JA
No affiliation info available
Kim PH
No affiliation info available
Ha KS
No affiliation info available

MeSH

Apoptosis Regulatory ProteinsBiosensing TechniquesGTP-Binding ProteinsGlutathioneGlutathione TransferaseGoldHumansIntracellular Signaling Peptides and ProteinsProtein Array AnalysisProtein Interaction MappingProtein Serine-Threonine KinasesRecombinant Fusion ProteinsSurface Plasmon ResonanceTransglutaminasesp21-Activated Kinasesrac1 GTP-Binding ProteinrhoA GTP-Binding Protein

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16402361

Citation

Jung, Jae-Wan, et al. "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." Proteomics, vol. 6, no. 4, 2006, pp. 1110-20.
Jung JW, Jung SH, Kim HS, et al. 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. Proteomics. 2006;6(4):1110-20.
Jung, J. W., Jung, S. H., Kim, H. S., Yuk, J. S., Park, J. B., Kim, Y. M., Han, J. A., Kim, P. H., & Ha, K. S. (2006). 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. Proteomics, 6(4), 1110-20.
Jung JW, et al. 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. Proteomics. 2006;6(4):1110-20. PubMed PMID: 16402361.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Jung,Jae-Wan, AU - Jung,Se-Hui, AU - Kim,Hyun-Soo, AU - Yuk,Jong Seol, AU - Park,Jae-Bong, AU - Kim,Young-Myeong, AU - Han,Jeong-A, AU - Kim,Pyung-Hyun, AU - Ha,Kwon-Soo, PY - 2006/1/13/pubmed PY - 2006/4/12/medline PY - 2006/1/13/entrez SP - 1110 EP - 20 JF - Proteomics JO - Proteomics VL - 6 IS - 4 N2 - 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. SN - 1615-9853 UR - https://www.unboundmedicine.com/medline/citation/16402361/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_ DB - PRIME DP - Unbound Medicine ER -