Preparation of recombinant AtBRI1-GC and functional testing in vitro.
(A) Testing of GC activity with an enzyme immunoassay. The control contains the reaction mixture without the substrate (10 µg recombinant protein in 50 mM Tris-HCl (pH 7.5), 2 mM isobutyl methylxanthine (IBMX), 5 mM Mg2+ and 5 mM Mn2+), the other columns represent cGMP generated in the presence of 1 mM GTP and either Mn2+ or Mg2+ after 20 min. The bar values represent the mean (+/−SEM). (B) Extracted mass chromatogram of m/z 344 [M-1]−1 ion of cGMP generated by 10 µg recombinant protein. The inset shows an SDS-PAGE of AtBRI1-GC expressed in E. coli BL21 (pLysS) (DE3) and purified with Ni-NTA agarose under denaturing conditions. Cleared lysate (lane 1), flow through (lane 2), first wash (lane 3), second wash (lane 4) and eluted recombinant protein (lane 5). ‘M’ is the molecular weight marker. (C) Extracted mass chromatogram of m/z 344 [M-1]−1 ion of the reaction mix without AtBRI1-GC. (D) Two superimposed extracted mass chromatogram of m/z 344 [M-1]−1 ion of cGMP generated by 10 µg recombinant protein after 5 and 20 min. respectively in the presence of 5 mM Mg2+. (Note that the sample was diluted 200 times as compared to the experiment presented in Fig. 2A). The left inset represents the mass of the peak in the chromatogram, the right inset is the calibration curve with 1.25, 10 and 50 fmoles on the column.