title: Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.
abstract: The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.
authors.affiliation: Department of Chemistry, University of Central Oklahoma, 100 N. University Dr., Edmund, Oklahoma, 73034, United States of America.
authors.forename: Yvonne D
authors.initials: YD
authors.lastname: Trigoso
authors.affiliation: Department of Chemistry, University of Central Oklahoma, 100 N. University Dr., Edmund, Oklahoma, 73034, United States of America.
authors.forename: Russell C
authors.initials: RC
authors.lastname: Evans
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma, 73019, United States of America.
authors.forename: William E
authors.initials: WE
authors.lastname: Karsten
authors.affiliation: Department of Chemistry, University of Central Oklahoma, 100 N. University Dr., Edmund, Oklahoma, 73034, United States of America.
authors.forename: Lilian
authors.initials: L
authors.lastname: Chooback
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D050746:Dihydrodipicolinate Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D050746:Dihydrodipicolinate Reductase
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