Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli.
Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
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Yang
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Hendrickson
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Kalman
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Crouch
D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012643:Selenium / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism*; D013696:Temperature; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D012645:Selenomethionine; D012260:Ribonucleases; D012643:Selenium