Expression of branching enzyme I of maize endosperm in Escherichia coli.
The gene encoding for mature branching enzyme (BE) I (BEI) of maize (Zea mays L.) endosperm has been expressed in Escherichia coli using the T7 promoter. The expressed BEI was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The recombinant enzyme showed properties very similar to those of BEI purified from developing maize endosperm with respect to branching amylose and amylopectin. This result confirmed our earlier report that maize endosperm BEI had a higher rate of branching amylose and a much lower rate (less than 10% of that of branching amylose) of branching amylopectin. This study also showed a great advantage in purifying BEI from the bacterial expression system rather than from developing maize endosperm. Most important, this study has established the system with which to study the structure-function relationships of the maize BEI using site-directed mutagenesis.
Department of Biochemistry, Michigan State University, East Lansing 48824.
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D015061:1,4-alpha-Glucan Branching Enzyme / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D015061:1,4-alpha-Glucan Branching Enzyme