title: High expression of glycogen-debranching enzyme in Escherichia coli and its competent purification method.
abstract: Glycogen-debranching enzyme (GDE) gene from Saccharomyces cerevisiae was cloned and expressed into Escherichia coli. A 99.3% homology was found between the nucleotide sequences of GDE gene harbored in the recombinant E. coli plasmid (pTrc99A) and the open reading frame (902039-906646 position) of the 4608-bp fragment of S. cerevisiae chromosome XVI. We investigated the best conditions for GDE expression. When the cultivation temperature of recombinant E. coli strains was lowered to 25 degrees C and the isopropyl-beta-d-thiogalactopyranoside (IPTG) concentration used for induction was decreased to as low as 0.02 mM, a total of about 33 mg of recombinant GDE can be isolated from a liter culture as estimated by amylo-1,6-glucosidase activity. Consecutively, we developed a new method for purifying GDE. The method requires only a single-step purification via beta-cyclodextrin-immobilized Sepharose 6B (beta-CD Sepharose 6B) affinity chromatography and renders a 90% recovery of the enzyme. Moreover, the purified recombinant GDE is a homogeneous protein and possesses the same characteristics as those of S. cerevisiae. With the highly expressed GDE in recombinant E. coli and a rapid and effective purification method, we successfully resolved the hurdle always faced for obtaining an ample amount of purified GDE. The availability of GDE, hence, may allow advancement on GDE studies and provide new prospects for GDE on biotechnological application.
authors.affiliation: Nara Prefectural Hospital, Hiramatsu, Nara City, Nara, 631-0846, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Nakayama
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: S
authors.initials: S
authors.lastname: Tabata
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013696:Temperature
chemical_list: D006004:Glycogen Debranching Enzyme System; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside