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 (C 85 and C 152), 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. 6mg/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.