Preparations of Bacillus pumilus secreted RNase: One enzyme or two?

Abstract

Immunochemical analysis of the following purified preparations of Bacillus pumilus RNase (binase) was carried out: industrially manufactured enzyme (Institute of Organic Synthesis, Riga, Latvia) and the enzymes isolated from the culture liquid of the native B. pumilus producer and from the Escherichia coli BL21 recombinant strain bearing the pGEMGX1/ent/Bi plasmid. Electrophoresis of all three samples of purified binase revealed two protein fractions with ribonuclease activity possessing molecular masses of ~12 and 25 kDa. The possible presence of binase II, a second secreted RNase, was ruled out. Both high and low molecular mass proteins interacted with binase specific antibodies in the immunoblotting reaction, which indicated their antigenic identity. The difference in molecular mass between these proteins indicated the pos sible presence of two forms of binase in solution, a monomer and a dimer.

Extracellular Ribonuclease fromBacillus licheniformis(Balifase), a New Member of the N1/T1 RNase Superfamily

“…Proteins were separated by SDS-PAGE [ 23 ] and transferred to a nitrocellulose membrane by Mini Trans-Blot cell (BioRad, USA). For the detection of proteins anti-binase antibodies were used [ 24 ]. Visualization of protein bands corresponding to RNase was performed using anti-rabbit IgG-POD secondary antibodies (Sigma-Aldrich, USA) and the LumiLight detection system (Roche Diagnostics, Switzerland).…”

The Cytotoxicity of RNase-Derived Peptides

“…Then, cells were fixed in a 4% paraformaldehyde solution for 15 min and permeabilized in 0.1% Triton-X100 solution in PBS for 10 min. Binase-treated cells were incubated overnight with anti-binase antibodies (1:500) at 4 °C [ 39 ]. Thereafter, the cells were washed three times in PBS containing 0.1% Tween for 10 min each and then were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-rabbit immunoglobulin G (IgG) (H+L) cross-adsorbed secondary antibody (Thermo Fisher Scientific, Inc., Waltham, MA, USA Cat.# 31584) at the concentration of 2 µg/mL in PBS containing 1% bovine serum albumin at room temperature for 45 min in the dark.…”

Three‐step procedure for preparation of pure Bacillus altitudinis ribonuclease

“…After separation by SDS/PAGE proteins were transferred to a nitrocellulose membrane by semi‐dry electroblotting. For the detection of proteins anti‐binase antibodies were isolated from rabbit blood as described earlier . The concentration of anti‐binase antibodies was 1 : 2000.…”