Expression and Purification of PF1684 Protein

To express the His-tagged PF1684 protein, we subcloned the PF1684 gene into the pET-23b expression vector by PCR using region-specific oligonucleotides as primers. These oligonucleotides were designed to provide PCR products with NdeI and XhoI sites at the 5'- and 3'- termini respectively. The resulting construct (termed pET-PF1684) contained the sequence coding for the PF1684 protein and six consecutive histidine residues (His-tag) at the C-terminus to be used as an affinity tag for purification. The nucleotide sequence of the insert was confirmed to be the original sequence. Recombinant PF1684 protein was overexpressed in E. coli strain BL21 (DE3) under the control of the phage T7 promoter. Protein was then extracted by sonication (30 s) in a buffer containing 20 mM Tris/HCl (pH 8.0), 500 mM NaCl, 5 mM imidazole and 0.1% Triton X-100. The extract was heat-treated at 80$\circ$ C for 15 min to denature endogenous E. coli proteins and then centrifuged at 12000 g for 10 min at 4$\circ$ C to separate the cell debris from the extract. The PF1684 protein was purified using a 5 ml Ni2+-Sepharose column according to the manufacturer's instructions (Novagen). The protein with peak RNA-binding activity was purified further by dialysis. SDS/PAGE showed that the purity of the PF1684 protein in the final preparation approached near homogeneity. 2.5 Determination of protein concentration Bio-RadŐs protein assay system was used to check the protein concentration based on the color change of Coomassie Brilliant Blue G-250 dye in response to various concentrations of protein. 2.6 DNA Sequencing The sequencing reaction was performed by PCR. The PCR product was then purified by chromatography (Centri-Sep) then sequenced by the sequencer (ABI 3100 Genetic Analyzer).