Fig 2. Recombinant Mimivirus L375 hydrolyzes the mRNA cap.

(A) An MBP-L375 fusion protein containing a C-terminal 10X histidine epitope tag (MBP-L375-HIS₁₀) was synthesized in Escherichia coli and purified by affinity chromatography through successive amylose and nickel-nitrilotriacetic acid columns. The purified MBP-L375-HIS₁₀ protein was separated by SDS/PAGE and visualized by Coomassie blue staining. The locations of the protein mass standards (in kDa) are labeled on the left side of the gel and the ~87 kDa MBP-L375-HIS₁₀ protein is denoted on the right. (B) MBP-L375-HIS₁₀ (80 ng) and 0.02 pmol ³²P-cap-labeled actin RNA were added to decapping buffer and incubated at 37°C for 30 min. After the incubation, an aliquot of the reaction was treated with 2 U of nucleoside diphosphate kinase (NDPK) in the presence of 1 mM ATP at 37°C for 30 min to convert nucleoside diphosphates into nucleoside triphosphates. The products of the reaction were separated on PEI-cellulose TLC plates in 0.75 M LiCl and the radioactive signals were visualized by autoradiography. Non-radioactive m⁷GDP and m⁷GTP standards were run in parallel and detected by UV shadowing; the migration of these standards is indicated on the right.