Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato ( Solanum lycopersicum) and its use to purify a vacuolar invertase

Abstract

Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank ( www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RT–PCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg −1) and efficiently degraded saccharose ( K m = 6.4 mM, V max = 2.9 μmol saccharose min −1 and k c at = 7.25 × 10 3 s −1 at pH 4.9 and 37 °C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDS–PAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH 2-terminal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated.