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

Published on Aug 1, 1986in Journal of Crystal Growth1.573
· DOI :10.1016/0022-0248(86)90172-7
Richard Giegé54
Estimated H-index: 54
(CNRS: Centre national de la recherche scientifique),
A. C. Dock5
Estimated H-index: 5
(CNRS: Centre national de la recherche scientifique)
+ 3 AuthorsDino Moras85
Estimated H-index: 85
(CNRS: Centre national de la recherche scientifique)
Abstract
Abstract In structural biology, the crystallization of the macromolecules often represents the most challenging step. Beside classical factors which determine the solubility of macromolecules, purity of compounds is another major parameter governing crystal growth. With aminoacyl-tRNA synthetases and transfer ribonucleic acids as examples, it will be shown that molecules to be crystallized not only have to be pure in terms of contaminating molecules, but also in terms of sequence integrity and conformational homogeneity. A chromatographic method based on salting-out of proteins or nucleic acids on Sepharose 4B gels and back-solubilization with inverse salt gradients will be discussed in the light of crystal growth experiments.
  • References (42)
  • Citations (72)
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References42
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Cited By72
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#1Arayik Martirosyan (UHH: University of Hamburg)
#2L.J. DeLucas (The Aerospace Corporation)H-Index: 1
Last. Christian Betzel (UHH: University of Hamburg)H-Index: 39
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1 CitationsSource
#1Catherine Florentz (UDS: University of Strasbourg)H-Index: 43
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#1Lata GovadaH-Index: 13
#2Naomi E. ChayenH-Index: 33
Anyone who has ever attempted to crystallise a protein or other biological macromolecule has encountered at least one, if not all of the following scenarios: No crystals at all, tiny low quality crystals; phase separation; amorphous precipitate and the most frustrating; large, beautiful crystals that do not diffract at all. In this paper we review a number of simple ways to overcome such problems, which have worked well in our hands and in other laboratories. It brings together information that ...
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#1Bernard LorberH-Index: 1
Pure and homogeneous biological macromolecules (i.e. proteins, nucleic acids, protein-protein or protein-nucleic acid complexes, and functional assemblies such as ribosomes and viruses) are the key for consistent and reliable biochemical and biophysical measurements, as well as for reproducible crystallizations, best crystal diffraction properties, and exploitable electron microscopy images. Highlights: Pure and homogeneous macromolecules are the key for the best experimental results; They warra...
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#1Jiong Chen (HAUST: Henan University of Science and Technology)H-Index: 1
#2Wei Feng (HAUST: Henan University of Science and Technology)H-Index: 1
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Abstract 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17β-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17β-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein...
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#1Mark L. Brader (University of Auckland)H-Index: 13
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Abstract Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why no...
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#1Alexander McPherson (UCI: University of California, Irvine)H-Index: 51
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Over the past 20 years a variety of technological advances in X-ray crystallography have shortened the time required to determine the structures of large macromolecules (i.e., proteins and nucleic acids) from several years to several weeks or days. However, one of the remaining challenges is the ability to produce diffraction-quality crystals suitable for a detailed structural analysis. Although the development of automated crystallization systems combined with protein engineering (site-directed...
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#1Richard Giegé (CNRS: Centre national de la recherche scientifique)H-Index: 54
Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both ‘practical’ and ‘basic’ science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X-ray crystallo...
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Transfer RNAs (tRNA) are small RNAs that provide the interface between DNA and ribosome-dependent protein synthesis, besides being involved in many other cellular processes. They interact with an impressive number of small molecule and macromolecular ligands and show structural and functional plasticity far to be deciphered. Here it is shown how tRNA biology was (and still is) idea and technology-driven and why crystallogenesis was at the heart of this science/technology interplay. Thus the ques...
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#1Alaa Adawy (Radboud University Nijmegen)H-Index: 6
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Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. T...
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