Molecular Cloning of a cDNA Encoding Granule-bound Starch Synthase I from Kidney Bean Seeds and Expression in Escherichia coli

Abstract

The major protein associated with starch granules from kidney bean (Phaseolus vulgaris L .) seeds is a granule-bound starch synthase I (designated PvGBSSI) with the molecular mass of 60 kDa. To analyze the nature of native granule-bound starch synthase I (GBSSI) enzyme, not the enzymes solubilized from starch granules, a study was undertaken to identify the GBSSI sequence expressed during seed development and to characterize the enzymatic properties of the coded recombinant enzyme . A cDNA clone (designated pvgbssl) was isolated from a cDNA library of kidney bean immature seeds . The predicted primary sequence of mature PvGBSSI displayed significant identity (66-81 %) to those of other GBSSI members . Analyses of Northern blot and starch granule proteins revealed that both transcript and protein for PvGBSSI showed maximum levels at the late to mature stages of seed maturation . To investigate enzymatic properties, recombinant PvGBSSI (rPvGBSSI) was purified from Escherichia coil as a single band of protein on SDS-polyacrylamide gel by one-step column chromatography. The activity of rPvGBSSI was stimulated 1.7-fold in the presence of 0.25 M citrate. The affinity for amylopectin with 0.25 M citrate was much higher than for that without citrate, while the maxim um velocities were constant with or without citrate .