title: Overproduction of Thermus sp. Strain T2 beta-galactosidase in Escherichia coli and preparation by using tailor-made metal chelate supports.
abstract: A novel thermostable chimeric beta-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the beta-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the beta-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports.
authors.affiliation: Department of Microbiology, Instituto de Fermentaciones Industriales, Madrid, Spain.
authors.forename: Benevides C C
authors.initials: BC
authors.lastname: Pessela
authors.forename: Alejandro
authors.initials: A
authors.lastname: Vian
authors.forename: César
authors.initials: C
authors.lastname: Mateo
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: José L
authors.initials: JL
authors.lastname: García
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.forename: Alfonso V
authors.initials: AV
authors.lastname: Carrascosa
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D001483:Base Sequence; D002614:Chelating Agents; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006639:Histidine; D007477:Ions; D019216:Metals, Heavy; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013824:Thermus / Q000201:enzymology* / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D007477:Ions; D019216:Metals, Heavy; D011993:Recombinant Fusion Proteins; D006639:Histidine; D001616:beta-Galactosidase