title: High-level expression of Trypanosoma brucei fructose-1,6-bisphosphate aldolase in Escherichia coli and purification of the enzyme.
abstract: A procedure has been developed for high-level expression of Trypanosoma brucei fructose-bisphosphate aldolase in Escherichia coli. Therefore, a specific restriction site was introduced by mutagenesis at the front of the gene, enabling its ligation in an expression plasmid, immediately downstream of the regulatory sequences. Growth conditions were established for production of high amounts of soluble and active enzyme. Aldolase was purified to near-homogeneity from the soluble fraction of the bacterial lysate by nuclease treatment, differential precipitation steps, and passage over a CM-Sepharose column. From a 1-liter culture of E. coli cells, 60-120 mg of purified protein that is essentially indistinguishable in physicochemical and kinetic properties and in stability from the enzyme purified from trypanosomes grown in infected laboratory animals was reproducibly obtained.
authors.affiliation: Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Chevalier
authors.forename: M
authors.initials: M
authors.lastname: Callens
authors.forename: P A
authors.initials: PA
authors.lastname: Michels
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase