title: One-step purification of soluble recombinant human 6-phosphogluconate dehydrogenase from Escherichia coli.
abstract: 6-Phosphogluconate dehydrogenase (6PGD), the third enzyme in the pentose phosphate pathway, was recently identified as a novel target in human lung cancer. In this report, we present an expression and purification scheme of recombinant human 6PGD from Escherichia coli. Using a DE3 derivative strain expressing tRNAs for seven rare codons in E. coli called Rosetta2 (DE3), a large quantity of soluble human 6PGD can be expressed with an N-terminal histidine tag and purified by a one-step purification procedure to near homogeneity without denaturants or refolding. Three to seven milligrams of purified protein could be obtained from 100 ml of culture. This recombinant human 6PGD follows classic Michaelis-Menton saturation kinetics with respect to both substrates NADP(+) and 6-phosphogluconate. The respective k(cat) and K(m) were comparable to those of 6PGDs purified from mammalian tissues. Using this purified 6PGD enzyme, we devised an endpoint colorimetric assay suitable for high-throughput screening for human 6PGD inhibitors.
authors.affiliation: Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, United States. Electronic address: cchan@bidmc.harvard.edu.
authors.forename: Barden
authors.initials: B
authors.lastname: Chan
authors.forename: Vikas P
authors.initials: VP
authors.lastname: Sukhatme
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005942:Gluconates / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D005942:Gluconates; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid
keywords: 6-Phosphogluconate dehydrogenase; Histidine-tag purification system; Pentose phosphate pathway