Urate Oxidase Purification by Salting-in Crystallization : Towards an Alternative to Chromatography

Background: Rasburicase (FasturtecH or ElitekH, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions

Multilevel Modular Mesocrystalline Organization in Red Coral

International audienceBiominerals can achieve complex shapes as aggregates of crystalline building blocks. In the red coral skeleton, we observe that these building blocks are arranged into eight hierarchical levels of similarly (but not identically) oriented modules. The modules in each hierarchical level assemble into larger units that comprise the next higher level of the hierarchy, and consist themselves of smaller, oriented modules. EBSD and TEM studies show that the degree of crystallographic misorientation between the building blocks decreases with decreasing module size. We observe this organization down to a few nm. Thus, the transition from imperfect crystallographic order at mm scale to nearly perfect single crystalline domains at nm scale is progressive. The concept of 'mesocrystal' involves the three-dimensional crystallographic organization of nanoparticles into a highly ordered mesostructure. We add to this concept the notion of 'multilevel modularity'. This modularity has potential implications for the origin of complex biomineral shapes in nature. A multilevel modular organization with small intermodular misorientations combines a simple construction scheme, ruled by crystallographic laws, with the possibility of complex shapes. If the observations we have made on red coral extend to other biominerals, long-range crystallographic order and interfaces at all scales may be key to how some biominerals achieve complex shapes adapted to the environment in which they grow

Non-monotonic variation with salt concentration of the second virial coefficient in protein solutions

The osmotic virial coefficient $B_2$ of globular protein solutions is calculated as a function of added salt concentration at fixed pH by computer simulations of the ``primitive model''. The salt and counter-ions as well as a discrete charge pattern on the protein surface are explicitly incorporated. For parameters roughly corresponding to lysozyme, we find that $B_2$ first decreases with added salt concentration up to a threshold concentration, then increases to a maximum, and then decreases again upon further raising the ionic strength. Our studies demonstrate that the existence of a discrete charge pattern on the protein surface profoundly influences the effective interactions and that non-linear Poisson Boltzmann and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory fail for large ionic strength. The observed non-monotonicity of $B_2$ is compared to experiments. Implications for protein crystallization are discussed.Comment: 43 pages, including 17 figure

Colloidal approach analysis of Marseille Protein Database for protein crystallization stragegies.

We have created a new online crystallization database, the Marseille Protein Crystallization Database (MPCD) [Charles et al. 2006, Acta Crystallogr. D62, 1311-1318], that includes information on macromolecules (name, pI, MW, number of subunits), crystallization conditions, methods and additives used, in standardized and tabulated form. It is freely accessible via http:// www.crmcn.univ-mrs.fr/mpcd/ and allows users to choose their own crystallization parameters, to create tables for further physicochemical analysis, and to enter new protein and crystallization conditions to supplement this database. The MPCD has previously been analyzed by Charles et al., by examining first each parameter independently to distinguish relevant data (i.e., PH, pI, nature and percentage of polymer) from statistical data (i.e., temperature, nature of salt). In the present paper, we analyze the database in regard to scattering studies and crystal growth experiments performed on various model systems. Some general trends of crystallization parameters are highlighted in correlation with results obtained from the study both of macromolecular interactions in solution using scattering techniques and of crystallization mechanisms using optical microscopy, to give some simple guidelines for biocrystallization. The main result is the synergistic effect of salt and polyethylene glycol observed both on interactions in solution, measured by scattering techniques and from the MPCD analysis

Properties of macromolecular solutions relevant to crystallization: Use of new amphiphilic molecules for soluble and membrane protein crystallization.

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Properties of macromolecular solutions relevant to crystallization: Use of new amphiphilic molecules for soluble and membrane protein crystallization.

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Protein crystallization: Contribution of small angle X-ray scattering (SAXS)

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The Hofmeister effect as seen by SAXS in protein solutions.

Protein interactions in solution are conveniently analysed with small angle X-ray scattering (SAXS). Such an analysis has shown that the addition of monovalent salt induces an additional attraction between macromolecules. A differential effect of the anions is observed, which follows the order of the Hofmeister series. Such experimental observations should be helpful to test the adequacy of theoretical models

Salting-In Effects on Urate Oxidase Crystal Design

In this paper, solubility and interactions in solution of the recombinant urate oxidase from Aspergillus flavus, rasburicase, are studied both in the absence and in the presence of salt at a pH close to the pI. An intense salting-in effect is demonstrated first by an increased solubility when various salts are added. Thus, merely adding salt does not induce rasburicase crystallization. Second virial coefficient measurements also confirm this effect by exhibiting repulsive interactions over a large range of salt concentrations. Therefore, the salting-in effect enables the stabilization of rasburicase solution at high concentrations. Moreover, it enables crystals of improved size and habit to be grown when polymer is added to a solution of rasburicase concentrated with salt, or when salt is removed from it. We also show, with the example of high pressure macromolecular crystallography, that salt enables the stabilization of the desired polymorph under the highly concentrated polyethylene glycol conditions required by this technique