The role of purification in the crystallization of proteins and nucleic acids

Giegé, Richard; Dock, Anne-Catherine; Kern, Daniel; Lorber, Bernard; Thierry, J. C.; Moras, Dino

doi:10.1016/0022-0248(86)90172-7

Cited by 82 publications

(37 citation statements)

References 38 publications

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“…It is well known that protein purification is a multistage and difficult process and the purity that can be achieved is commonly around 95%. Since the protein solutions used for crystallization are sensitive to impurity content [10,11] which may comprise dust particles, another protein or even conformational heterogeneity between molecules of one protein, we suggest a way for obtaining of protein crystals from protein mixtures. In our experiment we have used deliberately mixed solutions at different concentration ratios of two otherwise readily crystallizing proteins -chicken egg-white lysozyme and apoferritin from horse spleen.…”

Section: Introductionmentioning

confidence: 99%

Sedimentation as a tool for crystallization from protein mixtures

Dimitrov¹,

Nanev²

2006

Cryst. Res. Technol.

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Crystals from apoferritin which is an iron-free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO 4 in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (~1,14µM/L) in 25mg/ml (~1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (~1:103 ) of two proteins is used to stress on the observed effect.

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Section: Introductionmentioning

confidence: 99%

Sedimentation as a tool for crystallization from protein mixtures

Dimitrov¹,

Nanev²

2006

Cryst. Res. Technol.

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“…Purity requires both the removal of other protein species and the elimination of heterogeneity within the macromolecule to be crystallized. 12 Structurally dissimilar impurities are more likely to be rejected by growing crystals. [13][14] However, they may interfere with the nucleation process and may cause increased twinning 15 and loss of faceted faces.…”

Section: Introductionmentioning

confidence: 99%

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Snell

Judge

Crawford

et al. 2001

Crystal Growth & Design

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Chicken egg-white lysozyme (CEWL) crystals were grown in microgravity and on the ground in the presence of various amounts of a naturally occurring lysozyme dimer impurity. No significant favorable differences in impurity incorporation between microgravity and ground crystal samples were observed. At low impurity concentration the microgravity crystals preferentially incorporated the dimer. The presence of the dimer in the crystallization solutions in microgravity reduced crystal size, increased mosaicity, and reduced the signal-to-noise ratio of the X-ray data. Microgravity samples proved more sensitive to impurity. Accurate indexing of the reflections proved critical to the X-ray analysis. The largest crystals with the best X-ray diffraction properties were grown from pure solution in microgravity.

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“…The buffer was 20 mM Mes/ KOH pH 6.8 containing 0.1 mM EDTA, 5 mM 2-mercaptoethanol and 0.05 mM iPr,P-F; the column was operated at 4°C with a flow rate of 4.6 ml/h. For further details of this type of chromatography see [28]. Threonyl-tRNA synthetase elutes at 26% ammonium sulphate (Fig.1).…”

Section: Trna Enzymes and Chemicalsmentioning

confidence: 99%

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Theobald

Springer

Grunberg‐Manago

et al. 1988

European Journal of Biochemistry

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The solution structure of Escherichiu coli tRNA$' (anticodon GGU) and the residues of this tRNA in contact with the a2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA:hr. The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNAAspp, tRNAPhe and tRNATrp, already mapped with these probes.The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNATh' shows some similarities to that of yeast tRNAPhe and mammalian tRNATrp, especially in the D-arm (positions 19 and 24) and with tRNATrp, at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNAThr, similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA:h' is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNAAspp, the main differences in reactivity concern phosphates 19, 24 and 50.Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNAPhe. A striking conformational feature of tRNATh' is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs.Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extraloop, and in the amino acid accepting region. The involvement of the anticodon of tRNA:h' in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.The understanding of the specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases requires a precise structural knowledge of a number of different tRNA/synthetase systems. With such information it is expected one will find the conformational differences and similarities existing between such different systems and reveal structural rules responsible for the recognition process. From the tRNA point of view, many experimental (see...

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The role of purification in the crystallization of proteins and nucleic acids

Cited by 82 publications

References 38 publications

Sedimentation as a tool for crystallization from protein mixtures

Sedimentation as a tool for crystallization from protein mixtures

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

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The role of purification in the crystallization of proteins and nucleic acids

Cited by 82 publications

References 38 publications

Sedimentation as a tool for crystallization from protein mixtures

Sedimentation as a tool for crystallization from protein mixtures

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

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Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase