Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Bayol, Alain; Dupin, P.; Böe, J.; Claudy, P.; Létoffé, J. M.

doi:10.1016/0301-4622(94)00150-i

Cited by 20 publications

(18 citation statements)

References 11 publications

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“…Consistent with data reported previously ( Aspergillus flavus uricase) [31], two thermal transitions (TM1 and TM2) were observed for each uricase. Terriglobus saanensis and Deinococcus radiodurans uricases exhibited a thermal transition (TM1) that was lower than desired and, as a result, these two uricases were eliminated from the pool of candidates (Table 3).…”

Section: Resultssupporting

confidence: 92%

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

Nyborg¹,

Ward²,

Zacco³

et al. 2016

PLoS ONE

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Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

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Section: Resultssupporting

confidence: 92%

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

Nyborg¹,

Ward²,

Zacco³

et al. 2016

PLoS ONE

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“…2 as a function of pH between pH 7.5 and 10.5. Below pH 7.5 and above pH 10.5, the protein is unstable (Bayol et al, 1995). A 2 is minimum between pH 7.5 and 8.0 and then sharply increases until pH 10.5.…”

Section: Ph and Salt Effects: Saxs Experimentsmentioning

confidence: 97%

X-ray scattering studies ofAspergillus flavusurate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins

Vivarès¹,

Bonneté²

2002

Acta Crystallogr D Biol Cryst

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The determination of the three-dimensional structures of biological macromolecules by X-ray diffraction generally requires large good-quality crystals, which are often dif®cult to obtain as crystal nucleation and growth depend upon a great number of physicochemical parameters. In the future, the emergence of structural genomic projects will require new and rapid methods to determine crystallization conditions. Until now, the prediction of crystallization conditions has essentially been based on the knowledge of interparticular interactions in solutions inferred from studies on small soluble proteins in the presence of salts. The present study, by smallangle X-ray scattering, of urate oxidase from Aspergillus avus, a homotetrameric enzyme of 128 kDa, allowed the extension of the results to the crystallization of large proteins in the presence of polyethylene glycol (PEG). The protein crystallization, the nucleation rate and the different morphological crystal shapes obtained were correlated with the second virial coef®cient (A 2 ), which was found to be in a restricted range at the low end of the`crystallization slot' proposed by George & Wilson [(1994). Acta Cryst. D50, 361± 365].

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“…La principale différente avec l'urate oxydase extractive est que la rasburicase n'est pas complexée à la 8-azaxanthine. Elle présente la même structure que l'urate oxydase d'Aspergillus Flavus, mais elle est plus pure (Bayol et al, 1995).…”

Section: Urate Oxydase Recombinante Ou Rasburicaseunclassified

Versatile Microfluidic Approach to Crystallization

Zhang

Ferté

Candoni

et al. 2015

Org. Process Res. Dev.

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A l'issue de la rédaction de cette thèse, je suis convaincue que la recherche est loin d'être un travail solitaire. En effet, je n'aurais jamais pu réaliser ce travail doctoral sans le soutien d'un grand nombre de personnes. Cette thèse a été effectuée au CINaM, centre interdisciplinaire de nanosciences de Marseille. Je remercie M. Claude Henry, ancien directeur du laboratoire, et M. Frédéric Fages, directeur actuel du laboratoire, pour m'avoir accueillie pendant trois ans et m'avoir permis de réaliser ce travail. Je tiens également à remercier M. Roger Morin, directeur du département « Sources et Sondes Ponctuelles », pour ses encouragements chaleureux, ses précieux conseils sur la recherche et son organisation dynamique de la communication interdisciplinaire au sein du département. Je remercie aussi M. Mossadek Talby, de m'avoir accueillie dans l'Ecole Doctorale Physique et Sciences de la Matière, et pour le suivi tout au long de ma thèse. J'exprime toute ma reconnaissance aux deux rapporteurs de ma thèse, M. Claude Sauter et M. Tom Leyssens qui ont pris sur leur temps précieux pour examiner mon travail. Je suis également très reconnaissante envers Mme Fabienne Espitalier, M. Denis Mangin et M. Michael Lynch, qui me font l'honneur de participer au jury de soutenance. Mes sentiments les plus sincères vont à mes deux directeurs de thèse, Mme Nadine Candoni et M. Stéphane Veesler, pour la confiance qu'ils m'ont accordée, leur grande disponibilité et leurs qualités humaines d'écoute et de compréhension tout au long de ce travail, et surtout pour toutes les heures qu'ils ont consacrées à la relecture de ma thèse. Un grand merci pour les opportunités qu'ils m'ont fournies pour participer aux congrès et aux formations, au cours desquelles j'ai découvert des pays comme l'Espagne, le Japon et l'Angleterre. Nadine et Stéphane, vous m'avez aussi changée personnellement: avec vous, j'ai pris goût aux friandises !... Je tiens à remercier M. Mehdi Lagaize, M. Thirou Bactivelane ainsi que M. Michel Audiffren de la société Anacrismat, pour toutes les assistances techniques sur le développement du système microfluidique. Je remercie également M. Vasile Heresanu pour les expériences de SAXS. Sans vous, je n'aurais pas pu avancer mes expériences. Je tiens à remercier toutes les personnes qui m'ont donné des conseils précieux pendant ma thèse. J'exprime ma gratitude à Mme. Zoubida Hammadi pour sa façon de 10 2.1.3

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Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Cited by 20 publications

References 11 publications

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

X-ray scattering studies ofAspergillus flavusurate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins

Versatile Microfluidic Approach to Crystallization

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