Analysis of Interaction between PHO4 and
PHO2 Protein
by Real Time BIA
XIA Zan-Xian and AO Shi-Zhou
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China
)
Abstract Applying real time
BIA(biomolecular interaction analysis), the interaction between yeast PHO4 and
PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor
chip via amino group. 5 μmol/L of recombinant PHO2 and PHO2 mutants, which were
fused with glutathione S-transferase, were injected, respectively. The mutant, whose
Ser 230 was changed into Asp (GST-2SD) showed high SPR (surface plasmon
resonance) signal, while wild type PHO2 and the mutant where Ser 230 was
changed into Ala (GST-2SA) did not. This result indicated that there was
interaction between PHO4 and GST-2SD proteins, and the phosphorylation of the
Ser 230 of PHO2 was essential. Five different concentration of GST-2SD (0.5, 0.75,
1.0, 1.5, 2.0μmol/L) were injected separately, and the interaction kinetics was
analyzed. The association rate constant is 2.4×104(mol/L)–1s–1,
dissociation rate constant is 3.5×10–5s–1, and
association constant is 6.9×108 (mol/L)–1.
Key Words Real time BIA;PHO4;PHO2 and
mutant;SPR;association and dissociation rate constant
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