A novel approach of protein immobilization for protein chips using an oligo-cysteine tag.
Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.
Ichihara
Teruhisa
T
Technical Research Laboratory, Toyo Kohan Company, Ltd., Higashi-toyoi, 1296, Kudamatsu, Yamaguchi 733-8611,Japan.
Akada
Junko K
JK
Kamei
Shuichi
S
Ohshiro
Saemi
S
Sato
Daisuke
D
Fujimoto
Masanori
M
Kuramitsu
Yasuhiro
Y
Nakamura
Kazuyuki
K
D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D050777:Stathmin / Q000378:metabolism
D017931:DNA Primers; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D050777:Stathmin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003545:Cysteine