Expression, purification and properties of recombinant E. coli ribonuclease III.
Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over-expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild-type E. coli cells. The recombinant RNase III showed maximal activity at 37 degrees C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild-type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 microM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37 degrees and 50 degrees C. Incubation of RNase III at 37 degrees C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 degrees C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild-type RNase III.
Srivastava
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Department of Internal Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA. Srivasta@visar.wustl.edu
Srivastava
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D002845:Chromatography / Q000379:methods; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D043244:Ribonuclease III
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli