Rnasealert lab test kit

In vitro hydrolysis activity was assessed spectroscopically in 20 mM Tris pH 7.5 and 150 mM NaCl buffer using 3 mM cCMP. The cCMP solution was incubated with 20 µM NmMafB2-CT2 in a final volume of 80 µl at room temperature. And then the absorbance at 296 nm was measured for 10 min. As a positive control, bovine pancreatic RNase A at a concentration of 20 µM was included in the hydrolysis activity assay. To examine the dependency of metal atoms, metal atoms at a concentration of 0.2 mM was included in the reaction buffer. RNase activity was measured by a fluorophotometer using an RNaseAlert Lab Test Kit (Invitrogen). The reaction mixtures in the final 50 µl volume containing 10 µg of protein were prepared in black 96-flat well plates. The fluorescence intensity was measured at 37 °C for 30 min using a Tecan Spark microplate reader (Tecan) with emission (Em) at 530 nm and excitation (Ex) at 485 nm with a 20 nm bandwidth for both wavelengths. As negative controls, BSA and RNase-free H₂O were also included. All measurement were repeated three times.

RnaseAlert lab test kit (Invitrogen) was used to assay RNase A activity of the protein sample. The reaction mix consist of 10 μl of RNase A substrate (20× dilution with RNase A buffer solution), 5 μl of protein sample from periplasmic fraction (1000×), and 85 μl of nuclease-free water. As a negative control, 5 μl of protein buffer or 5 μl of protein sample from periplasmic fraction of non RNase A producer strain was added instead into the reaction mix. The reaction mix was transferred into 96-well plate, and then incubated in the Clariostar plate reader for 1 h 37 °C. The RNase A substrate from the kit is a modified RNA nucleotide that emits a green fluorescence when it is degraded by RNase. The fluorescence activity of degraded substrate was recorded overtime during the incubation (RFU at 490/520 nm). RNase A specific activity was quantified by normalising the rate of substrate degradation (RFU/minute) with the amount of protein sample. All data were produced in biological triplicate.

The coding sequences of PhS₃‐RNase (without signal peptide) and PhS₃‐RNase (M) were separately cloned into the pCold‐TF vector (TaKaRa, Kusatsu, Japan). Relevant primer sequences used are listed in Table S1. Trigger Factor (TF) is a 48 kDa soluble tag located at the N‐terminus of His. The His‐fused proteins were expressed in Escherichia coli Trans BL21 (DE3) plysS (Transgen) at 16°C for 24 h at 180 rpm and purified using Ni Sepharose 6 Fast Flow beads (10249123; GE Healthcare, Waukesha, WI, USA) according to the manufacturer’s instructions. Protein concentration was determined by Bradford protein assays. The relative fluorescence units (RFU) of the recombinant proteins were monitored according to the manufacturer’s instructions of RNase Alert Lab Test Kit (Ambion) using Synergy 2 (Biotek, Winooski, VT, USA).