title: Immobilization of dihydrofolate reductase by engineered cysteine residue attached to its C-terminal end.
abstract: A cysteine residue with a short amino acid chain spacer was attached to the C-terminal end of mutant dihydrofolate reductase [DHFR(C152E)] by a recombinant DNA technique. By using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a SH reagent, it was determined that the reactivity of the SH group of the introduced C-terminal cysteine was much higher than that of cysteines with the wild-type DHFR (C85 and C152), probably due to an increase of the solvent accessible surface area of the SH group. By utilizing the increased reactivity of the SH group of the C-terminal cysteine, the engineered DHFR was effectively immobilized to a thiopropyl-Sepharose gel. The amount of immobilized DHFR was estimated to be ca. 6 mg/g of dried gel, and the activity of the bound DHFR was comparable to that of the free enzyme. Thus, the attachment of the cysteine residue to the C-terminal was extremely useful for immobilization of the enzyme.
authors.affiliation: National Institute of Bioscience and Human Technology, Ibaraki.
authors.forename: M
authors.initials: M
authors.lastname: Iwakura
authors.forename: T
authors.initials: T
authors.lastname: Kokubu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D012685:Sepharose; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
chemical_list: D004800:Enzymes, Immobilized; D013438:Sulfhydryl Compounds; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine