CRYSTAL GROWTH & DESIGN
Salting-In Effects on Urate Oxidase Crystal Design† Marion Giffard,‡ Nathalie Colloc’h,§ Natalie Ferte´,‡ Bertrand Castro,| and Franc¸oise Bonnete´*,‡ Centre Interdisciplinaire de Nanoscience de Marseille, CINaM-CNRS UPR 3118, Aix-Marseille UniVersite´, Parc Technologique et Scientifique de Luminy, Case 913, 13288 Marseille Cedex 09, France, CI-NAPS UMR 6232-UCBN-CNRS, GIP Cyceron, Bd Becquerel, BP5229, 14074 Caen cedex, France, and Sanofi-AVentis, 371 Rue du Professeur Blayac, 34184 Montpellier, France
2008 VOL. 8, NO. 12 4220–4226
ReceiVed June 26, 2008; ReVised Manuscript ReceiVed September 29, 2008
ABSTRACT: 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.
1. Introduction Crystallization is a solid-liquid interfacial process, used for formulation and purification in industry1,2 and to grow highquality crystals for structure determination in crystallography. Often, in biocrystallography, proteins are too scarce and are mainly crystallized by trial-and-error methods, using nanovolume crystallization robots. However, pharmaceutical processes require large amounts of protein to determine phase diagrams, and in this context knowledge of solubility is essential to control polymorphism, nucleation and yield.3-5 Solubility, that is, the concentration at which the protein solution is in equilibrium with the crystal phase, varies with physicochemical conditions (pH, temperature, additives). Its variations are also commonly studied to gain insight into the process of protein crystallization according to the protein properties in solution (isoelectric point, molecular weight, stability, etc.). An additional approach to solubility measurement is the study of weak interaction forces between proteins in solution.6-12 Most articles report that addition of salt decreases solubility.13,14 This is confirmed by the fact that salt induces attractive interactions through charge screening.10 This effect, known as salting-out,15 is generally observed at medium and high salt concentrations.16 At low ionic strength the opposite effect is expected,17 that is, salting-in, where solubility increases with addition of salt. However, salting-in has only sporadically been reported.17-20 One reason is that few protein solubility curves have been characterized. A second reason is that some proteins do not exhibit salting-in at all, for instance lysozyme.21 A third reason is that only physicochemical conditions leading to crystallization are reported in the crystallography literature; so a phenomenon increasing protein solubility and directly dissolving crystals, even if observed, is not always mentioned. † Part of the special issue (Vol 8, issue 12) on the 12th International Conference on the Crystallization of Biological Macromolecules, Cancun, Mexico, May 6-9, 2008. * To whom correspondence should be addressed. E-mail: bonnete@ cinam.univ-mrs.fr; phone: +33 (0) 662 922 839; fax: +33 (0) 491 418 916. ‡ CINaM-CNRS UPR 3118, Aix-Marseille Universite´. § CI-NAPS UMR 6232-UCBN-CNRS. | Sanofi-Aventis.
Among the different proteins available for crystallization studies,13,22-26 urate oxidase has been studied widely with respect to its crystallization with polyethylene glycol.22,27-29 It has also been shown that addition of salt does not lead directly to any solid phase. Indeed, the usual salting-out effect has been predicted via weak interaction forces at a pH far from the pI, but is not intense enough to induce crystallization, whereas at a pH closer to the pI interactions between these proteins are more consistent with salting-in.30 The urate oxidase (uricase, EC 1.7.3.3, uox) gene, inactive in humans and higher primates, encodes for an enzyme which catalyzes the oxidation of uric acid to allantoin, an inactive and soluble metabolite. Urate oxidase is used as a protein drug to reduce toxic uric acid accumulation and to treat the hyperuricemic disorders occurring during chemotherapy; it is produced, purified, and made commercially available by Sanofi-Aventis. Initially, urate oxidase was extracted from Aspergillus flaVus, but now the drug is produced by genetic engineering and the recombinant urate oxidase is called rasburicase. Rasburicase has been shown to be less soluble than extractive urate oxidase under the same solvent conditions31 (probably due to the higher purity of the recombinant enzyme and to the presence of a cysteine adduct on the extractive urate oxidase32), raising the question of rasburicase stability in solution and of additives that can be used to increase its solubility. Here we first report results on rasburicase solution stabilization by addition of salt. Then we show how this effect can be used to improve crystal growth and design, in particular with X-ray crystallography under high pressure.
2. Experimental Section 2.1. Solutions. The recombinant urate oxidase (rasburicase) from A. flaVus expressed in Saccharomyces cereVisiae was supplied purified by Sanofi-Aventis without inhibitor in a phosphate buffer. Rasburicase was placed in 50 mM Tris buffer pH 8, using gel filtration ¨ KTA basic system chromatography on Superdex S200PG with an A and concentrated by ultrafiltration on an Amicon cell. 8-Azaxanthine (Sigma-Aldrich) was incubated when needed with the rasburicase solution and any excess was removed using gel filtration chromatography as described above. The rasburicase stock solution was kept at
10.1021/cg800679v CCC: $40.75 2008 American Chemical Society Published on Web 11/08/2008
Salting-In Effects on Urate Oxidase Crystal Design 3 mg/mL in a cold room and concentrated when needed. One molar stock solutions of sodium chloride, potassium chloride ammonium chloride and 500 mM magnesium chloride, calcium chloride, sodium sulfate, ammonium sulfate (Sigma-Aldrich) were prepared by dilution of the appropriate amount of salt in 50 mM Tris buffer pH 8. A solution of 40% w/v of PEG 8000 in 50 mM Tris buffer pH 8 was prepared from a 50% w/v solution (Hampton Research). All salt and rasburicase solutions for crystallization trials were filtered on 0.22 µm MILLIPORE filters. 2.2. Isoelectric Focusing. All reagents were purchased from BioRad: ReadyStrip IPG Strips narrow range 5-8 17 cm, 10× anode buffer, 7 mM phosphoric acid; 10× cathode buffer 20 mM lysine, 20 mM arginine; sample buffer 50% glycerol; gel stain Coomassie R-250/ Crocein Scarlet and protein standard consisting of a mixture of nine native proteins with isoelectric points ranging from 4.45 to 9.6 (cytochrome c, lentil lectin, human hemoglobin C and A, equine myoglobin, human and bovine carbonic anhydrase, beta lactoglobulin B and phycocyanin). Twenty microliters of each 1 mg/mL sample were uploaded into the wells of the gel and power was applied (100 V for the first hour then 200 V for 1 h and finally 300 V for 30 min). The gel was then bathed in the Coomassie staining solution for 30 min and washed overnight in a destaining solution (450 mL of 100% methanol, 450 mL of deionized water, 100 mL of glacial acetic acid). 2.3. Small Angle X-ray Scattering Experiments. Small angle X-ray scattering (SAXS) experiments were performed at HASYLAB (EMBL, DESY, Hamburg) where measurements can be performed rapidly, typically in 120 s. The X-ray beam was monochromatized (0.15 nm, horizontal focusing triangular Si (111) asymmetric cut 7°) and focused with a rhodium-coated flat mirror on Zerodur substrate with gravimetrical bending. Data were collected using a two-dimensional Mar345 image plate with online readout. Several series of experiments were performed. The sample-to-detector distance was 2.4 m leading to an average recorded s-range s ) 0.08-4.5 nm-1. The solution experiments were performed with a 50 µL quartz cell operated in vacuum. Second virial coefficients were calculated from scattered intensity, as previously described.33 2.4. Crystallization Setup. The crystallization trials prior to crystal growth studies were performed in an air-conditioned room (20 °C) using the microbatch and dialysis techniques. For dialysis, the protein solution was placed in a 10 µL button covered with a 3500 Da cutoff membrane purchased from Spectra/Por prior to equilibration versus 500 µL of deionized water for one day. For microbatch, droplets were prepared by mixing the concentrated and purified protein solution with the precipitant agent (PEG 8000 40%) and, when needed, buffer (Tris 50 mM) and salt (NaCl or KCl 1 M). The droplets were pipetted under a layer of paraffin oil in a 72-well microbatch plate (paraffin oil and plates from Hampton Research). The final volumes were 10 µL. To favor growth, droplets were seeded with a crystallized solution at higher protein concentration under identical precipitant conditions. When no crystals were obtained at a given precipitant concentration, seeds obtained under higher precipitant conditions were used. Crystals were observed with a TE 200 microscope (Nikon) and photographs were taken at 200× enlargement using the Replay software (Microvision), one day after the droplet preparation and seeding. The crystal growth studies were performed in Peltier controlledtemperature devices using small glass cells (
