Ribonucleotide Reductase Enzyme Purification

Wt-α2 (2500 nmol/min/mg) and wt-β2 (1.2 Y₁₂₂•/β2, 7600 nmol/min/mg) were expressed from pMJ1-nrdA and pTB2-nrdB, respectively, and purified as previously described.^{22 (link),23 (link)} Y₇₃₁NH₂Y-α2 and Y₇₃₀NH₂Y-α2 were co-expressed from pTrc-nrdA-TAG₇₃₁ or pTrc-nrdA-TAG₇₃₀ and pAC-NH₂Y, and purified as described.^{17 (link)} All α2 proteins were pre-reduced prior to use.^{17 (link)} E. coli thioredoxin (TR, 40 U/mg) and thioredoxin reductase (TRR, 1400 U/mg) were isolated as described.^{24 (link),25 (link)} 2´-Azido-2´-deoxycytidine 5´-diphosphate (N₃CDP) was synthesized from uridine by known procedures.^{26 (link),27 (link)} [5-³H]-CDP was purchased from ViTrax (Placentia, CA). Nucleotide primers were purchased from Invitrogen, Pfu Ultra II polymerase from Stratagene, and restriction enzymes from New England Biolabs. Assay buffer consists of 50 mM Hepes, 1 mM EDTA, and 15 mM MgSO₄, pH 7.6. Generation of pTrc-nrdB, pTrc-nrdB(TAG₃₅₆), pET-nrdA(wt), pET-nrdA(TAG₇₃₀), and pEVOL-NH₂Y, and expression and purification of N-Strep-Y₇₃₀NH₂Y-α2, N-Strep-Y₃₅₆NH₂Y-β2, and (His)₆-Y₃₅₆NH₂Y-β2 are described in detail in the Supporting Information (SI).