- Email: [email protected]
Crystallization of the ColE1 rop protein
J. Mol. Biol. (1983) 170, 1059-1060
Crystallization of the ColE1 Rop Protein Preliminary crystallographic data are given for Rop, a protein involved in the control of replication of plasmids of the ColEI family.
Plasmids related to CotE1 (e.g. ColK, pBR322) are present in Escherichia coli cells at a level of about 15 plasmid copies per genome and share a common, highly conserved, replication origin region. Replication of the closed-circular doublestranded DNA requires a plasmid-encoded RNA primer but will proceed in the absence of host protein synthesis. Twigg & Sherratt (1980) reported that deletion of a DNA restriction fragment 500 nucleotides downstream of the origin of replication resulted in increased plasmid copy number. This region was later shown to code for a 63 amino acid protein called Rop (for Repressor Of Primer), which reduces the rate of replication by inhibiting primer formation (Cesareni et al., 1982; Som & Tomizawa, 1983). We report here the crystallization of this protein in its native state. The appropriate DNA from pBR322 was cloned into plasmid pLC28 (Remaut et al., 1981), under the control of the 2 pL promoter. Expression of the gene in a cell line containing )[ prophage having a thermosensitive repressor enabled production of a protein whose subunit molecular weight on polyacrylamide gels is around 6000. This is compatible with the l%op molecular weight of 7148 deduced from the DNA sequence. Rop protein was isolated at about 90~/o purity by ion-exchange chromatography and gel filtration as described by Lacatena et al. (1983). Gel filtration in 0-1 M-salt indicates a molecular weight of 17,000 to 18,000. The protein is thus most probably dimeric under physiological conditions (see below). Repeated crystallization near the isoelectric pH value of 6 gives pure Rop as assessed by gel electrophoresis and amino acid analysis. Up to 30 mg of pure protein may be obtained from 50 g of cell paste. Crystals of Rop may be grown from solutions containing ammonium sulphate, polyethylene glycol or organic solvents. Dialysis of Rop solutions concentrated to over 1 mg/ml against ammonium sulphate solutions at around pH 6 gives, after several days, sheaves of small needles suitable for protein purification but not suitable for X-ray studies. By contrast, needle crystals up to 1 mm long form rapidly in the presence of polyethylene glycol, citrate and magnesium at pH 6. Typically, a 5 mg/ml solution of Rop in 20 mM-imidazole buffer at pH 7-4 is made 5~/o (w/v) in polyethylene glycol 6000. Magnesium sulphate is then added to a final concentration of 100 mM and sodium citrate buffer to a final concentration of 50 mM and pH 6. Needle crystals form at once. Crystallization trials reaching the same endpoint in different ways have not yet given crystals of more than about 0-1 mM diameter. It may be noted that these crystals will grow on the addition of 1059 0022-2836/83/321059-02 $03.00/0 © 1983 AcademicPress Inc. (London) Ltd.
10{10
D. W. B A N N E R ,
G. C E S A R E N I
AND
D. T S E R N O G L O U
distilled water to the precipitate formed when the citrate or magnesium concentration is too high. Large crystals suitable for high-resolution X-ray studies m a y be obtained by taking 5 mg/ml Rop solutions in low concentrations of p H 7-4 buffers and making them about 50~/o in a range of organic solvents. Vapour diffusion against, for instance, ethanol, methanol or acetone gives well-formed, lozenge-shaped crystals. The most successful procedure to date is simply to layer carefully an equal volume of 2-methyl-2,4-pentane diol over about I0 pl of Rop solution in a glass capillary held somewhat away from the vertical (cf. Salemme, 1972). Crystals of up to 1-2 m m by 0.4 m m by 0.4 m m grow in one to two weeks. They diffract to beyond 2 A and are not unusually sensitive to X-irradiation. The crystals are monoclinic space group C2, with cell dimensions a = 57.3 A, b = 40.2 A, c = 27.2 A, fl = 103 °. The volume per unit molecular weight is thus 2,14 A3/dalton on the assumption of one monomer per asymmetric unit (4 per cell). This value is typical for proteins (Matthews, 1968). The unit cell m a y be considered to contain two symmetrical dimers. This would then be consistent with a dimeric form in solution. We are pursuing high-resolution X - r a y diffraction studies on this crystal form. European Molecular Biology Laboratory Postfach 10.2209 69 Heidelberg, BRD
DAVID W. BANNER GIANNI CESARENI DEMETRIUS TSERNOOLOU
Received 9 August 1983 REFERENCES Cesareni, G., Muesing, M. A. & Polisky B, (1982). Proc. Nat. Acad. Sci, U.S.A. 79, 63136317. Lacatena, R. M., Banner, D. W. & Cesareni, G. (1983). In Mechanisms of DNA Replication and Recombination (Cozzarelli, ed.), Alan R. Liss, Inc., New York. In the press. Matthews, B. W. (1968). J. Mol. Biol. 33,491-497. Remaut, E., Straussens, P. & Fiers, W. (1981). Gene, 15, 81-93. Salemme, F. R. (1972). Arch. Biochem. Biophys. 151,533-539. Som, T. & Tomizawa, J.-I. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 3232-3236. Twigg, A. J. & Sherratt, D. (1980). Nature (London), 283, 216-218. Edited by A. Klug
Crystallization of the ColE1 Rop Protein Preliminary crystallographic data are given for Rop, a protein involved in the control of replication of plasmids of the ColEI family.
Plasmids related to CotE1 (e.g. ColK, pBR322) are present in Escherichia coli cells at a level of about 15 plasmid copies per genome and share a common, highly conserved, replication origin region. Replication of the closed-circular doublestranded DNA requires a plasmid-encoded RNA primer but will proceed in the absence of host protein synthesis. Twigg & Sherratt (1980) reported that deletion of a DNA restriction fragment 500 nucleotides downstream of the origin of replication resulted in increased plasmid copy number. This region was later shown to code for a 63 amino acid protein called Rop (for Repressor Of Primer), which reduces the rate of replication by inhibiting primer formation (Cesareni et al., 1982; Som & Tomizawa, 1983). We report here the crystallization of this protein in its native state. The appropriate DNA from pBR322 was cloned into plasmid pLC28 (Remaut et al., 1981), under the control of the 2 pL promoter. Expression of the gene in a cell line containing )[ prophage having a thermosensitive repressor enabled production of a protein whose subunit molecular weight on polyacrylamide gels is around 6000. This is compatible with the l%op molecular weight of 7148 deduced from the DNA sequence. Rop protein was isolated at about 90~/o purity by ion-exchange chromatography and gel filtration as described by Lacatena et al. (1983). Gel filtration in 0-1 M-salt indicates a molecular weight of 17,000 to 18,000. The protein is thus most probably dimeric under physiological conditions (see below). Repeated crystallization near the isoelectric pH value of 6 gives pure Rop as assessed by gel electrophoresis and amino acid analysis. Up to 30 mg of pure protein may be obtained from 50 g of cell paste. Crystals of Rop may be grown from solutions containing ammonium sulphate, polyethylene glycol or organic solvents. Dialysis of Rop solutions concentrated to over 1 mg/ml against ammonium sulphate solutions at around pH 6 gives, after several days, sheaves of small needles suitable for protein purification but not suitable for X-ray studies. By contrast, needle crystals up to 1 mm long form rapidly in the presence of polyethylene glycol, citrate and magnesium at pH 6. Typically, a 5 mg/ml solution of Rop in 20 mM-imidazole buffer at pH 7-4 is made 5~/o (w/v) in polyethylene glycol 6000. Magnesium sulphate is then added to a final concentration of 100 mM and sodium citrate buffer to a final concentration of 50 mM and pH 6. Needle crystals form at once. Crystallization trials reaching the same endpoint in different ways have not yet given crystals of more than about 0-1 mM diameter. It may be noted that these crystals will grow on the addition of 1059 0022-2836/83/321059-02 $03.00/0 © 1983 AcademicPress Inc. (London) Ltd.
10{10
D. W. B A N N E R ,
G. C E S A R E N I
AND
D. T S E R N O G L O U
distilled water to the precipitate formed when the citrate or magnesium concentration is too high. Large crystals suitable for high-resolution X-ray studies m a y be obtained by taking 5 mg/ml Rop solutions in low concentrations of p H 7-4 buffers and making them about 50~/o in a range of organic solvents. Vapour diffusion against, for instance, ethanol, methanol or acetone gives well-formed, lozenge-shaped crystals. The most successful procedure to date is simply to layer carefully an equal volume of 2-methyl-2,4-pentane diol over about I0 pl of Rop solution in a glass capillary held somewhat away from the vertical (cf. Salemme, 1972). Crystals of up to 1-2 m m by 0.4 m m by 0.4 m m grow in one to two weeks. They diffract to beyond 2 A and are not unusually sensitive to X-irradiation. The crystals are monoclinic space group C2, with cell dimensions a = 57.3 A, b = 40.2 A, c = 27.2 A, fl = 103 °. The volume per unit molecular weight is thus 2,14 A3/dalton on the assumption of one monomer per asymmetric unit (4 per cell). This value is typical for proteins (Matthews, 1968). The unit cell m a y be considered to contain two symmetrical dimers. This would then be consistent with a dimeric form in solution. We are pursuing high-resolution X - r a y diffraction studies on this crystal form. European Molecular Biology Laboratory Postfach 10.2209 69 Heidelberg, BRD
DAVID W. BANNER GIANNI CESARENI DEMETRIUS TSERNOOLOU
Received 9 August 1983 REFERENCES Cesareni, G., Muesing, M. A. & Polisky B, (1982). Proc. Nat. Acad. Sci, U.S.A. 79, 63136317. Lacatena, R. M., Banner, D. W. & Cesareni, G. (1983). In Mechanisms of DNA Replication and Recombination (Cozzarelli, ed.), Alan R. Liss, Inc., New York. In the press. Matthews, B. W. (1968). J. Mol. Biol. 33,491-497. Remaut, E., Straussens, P. & Fiers, W. (1981). Gene, 15, 81-93. Salemme, F. R. (1972). Arch. Biochem. Biophys. 151,533-539. Som, T. & Tomizawa, J.-I. (1983). Proc. Nat. Acad. Sci., U.S.A. 80, 3232-3236. Twigg, A. J. & Sherratt, D. (1980). Nature (London), 283, 216-218. Edited by A. Klug