A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.
Protein Expr Purif
; 75(2): 192-203, 2011 Feb.
Article
em En
| MEDLINE
| ID: mdl-20851186
ABSTRACT
Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Projetos de Pesquisa
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Proteínas Recombinantes
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Corpos de Inclusão
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Anafilatoxinas
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Fator Estimulador de Colônias de Granulócitos
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Interleucina-17
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Quimiocina CXCL12
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Quimiocina CXCL13
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Ensaios de Triagem em Larga Escala
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article