Search

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3-5 exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat) = 100-650 min(-1), K-M = 0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn2+). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn2+ being preferred over Mg2+ as cofactor, and was inhibited by Ni2+. The concentration dependency of Mn2+ was examined, giving K-Mn values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage1, 2; it is best known for its roles in insect flight3, 4 and the remarkable jumping ability of fleas5, 6 and spittle bugs7. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein8, 9 (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.