Expression of soluble and catalytically active plant (monocot) beta-glucosidases in E. coli.
Complementary DNAs encoding mature beta-glucosidase proteins Glu1 and Glu2 of maize were amplified by the polymerase chain reaction (PCR) and cloned into the expression vector pET21a. Both Glu1 and Glu2 isozymes were expressed in high yield ( approximately 3.8% of the total soluble protein and 32% of the total expressed protein) in E. coli. Recombinant enzymes were active on a variety of artificial and natural substrates at levels similar to those of their native counterparts isolated from maize seedlings. Western blot analysis confirmed that both recombinant isozymes were immunoreactive with maize anti-beta-glucosidase sera and their molecular sizes were identical to those of the native maize Glu1 and Glu2 isozymes. Zymogram assays in native gels revealed that recombinant enzymes had the same electrophoretic mobility and substrate specificity as their native counterparts.
Cicek
M
M
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0406, USA.
Esen
A
A
D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D002523:Edible Grain / Q000201:enzymology; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D001617:beta-Glucosidase