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.
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.
Jung
Jae-Wan
JW
Department of Molecular and Cellular Biochemistry, Kangwon National University College of Medicine, Chuncheon, Kangwon-Do, Korea.
Jung
Se-Hui
SH
Kim
Hyun-Soo
HS
Yuk
Jong Seol
JS
Park
Jae-Bong
JB
Kim
Young-Myeong
YM
Han
Jeong-A
JA
Kim
Pyung-Hyun
PH
Ha
Kwon-Soo
KS
D051017:Apoptosis Regulatory Proteins; D015374:Biosensing Techniques; D019204:GTP-Binding Proteins; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D040081:Protein Array Analysis; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D011503:Transglutaminases / Q000378:metabolism; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; C100494:RTKN protein, human; D011993:Recombinant Fusion Proteins; D006046:Gold; D011503:Transglutaminases; D005982:Glutathione Transferase; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D019204:GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D005978:Glutathione