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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
Author(s) -
Jung JaeWan,
Jung SeHui,
Kim HyunSoo,
Yuk Jong Seol,
Park JaeBong,
Kim YoungMyeong,
Han JeongA,
Kim PyungHyun,
Ha KwonSoo
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500371
Subject(s) - fusion protein , biosensor , surface plasmon resonance , rhoa , glutathione , chemistry , biochemistry , protein array analysis , green fluorescent protein , biophysics , rac1 , recombinant dna , microbiology and biotechnology , biology , enzyme , materials science , gene expression , nanotechnology , signal transduction , nanoparticle , gene , dna microarray
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‐p 21 ‐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 Ca 2+ ‐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 Ca 2+ ‐dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca 2+ 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.