Figure 3.

Recombinant AtCPT7 Enzyme Activity and Substrate Specificity.

(A) Purification of His₆-tagged AtCPT7 by Ni²⁺ affinity chromatography. Proteins were separated by SDS-PAGE and stained with Coomassie blue. For each protein, lane 1 was loaded with E. coli extract containing 10 µg of total protein and lane 2 with 2 µg of purified protein.

(B) Analysis of enzymatic reaction products. Recombinant AtCPT7 was incubated with ¹⁴C-IPP together with either FPP or GGPP and the dephosphorylated reaction products were resolved on reverse-phase silica gel 60-Å plates using an acetone/water (39:1) solvent system and developed by autoradiography. The position of C55 was determined based on the migration of authentic polyprenol standards of known size (C10–C120); the solvent front (SF) and origin (O) are also indicated.

(C) Recombinant AtCPT7 was assayed with various concentrations of either GPP or GGPP, and enzyme activity was determined by scintillation counting as described in Methods. Results are representative from experiments performed with three independent preparations of recombinant protein.