Figure 6.

Expression analysis of Sbe1c in E. coli. A, Schematic illustration of the expression vector pQE-SBEIc carrying sequences encoding mature SBEIc with His tag (black box) added at the amino-terminal end. B, Analysis of BE activity by iodine staining and phosphorylase a stimulation assay. BE activities were determined from the BE-positive strain DH5α and the BE-deficient strain KV832, transformed with plasmids indicated. Construct pREP4-cm expresses the Lac repressor and pQE30 is a cloning vector used for construction of pQE-SBEIc. The BE activity values and ses determined by the phosphorylase a stimulation assay (Hawker et al., 1974) are expressed as μmol Glc-1-P incorporated mg protein⁻¹ min⁻¹ and were determined from three separate experiments. C, SDS-PAGE and immunoblot analysis of recombinant wheat SBEIc produced in E. coli. Total cell extracts of noninduced and IPTG-induced cultures of the BE-deficient strain, KV832, harboring pREP4-cm and plasmid indicated were analyzed. The immunoblot analysis was done with antibodies prepared against wheat 87-kD SBEI. Migration of marker proteins revealed by amido black staining is shown to the right.