CO/bicarbonate modulates cone photoreceptor ROS-GC1 and restores its CORD6-linked catalytic activity.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO. Mechanistically, CO is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction.

Construction of an overproducing strain, purification, and biochemical characterization of the 6His-Eco29kI restriction endonuclease.

We constructed a strain of Escherichia coli overproducing 6His-tagged Eco29kI by placing the coding sequence under control of a strong bacteriophage T5 promoter. The yield of 6His-Eco29kI restriction endonuclease expression could be increased to about 20% of the total cellular protein, but inclusion bodies formed consisting of insoluble 6His-Eco29kI protein. We developed a fast and effective protocol for purification of the homogeneous enzyme from both soluble and insoluble fractions and established their identity by catalytic activity assay.