RESUMEN
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.
Asunto(s)
Anafilatoxinas/química , Anafilatoxinas/metabolismo , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Cuerpos de Inclusión/química , Interleucina-17/química , Interleucina-17/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proyectos de Investigación , Anafilatoxinas/genética , Anafilatoxinas/aislamiento & purificación , Bioensayo , Quimiocina CXCL12/genética , Quimiocina CXCL12/aislamiento & purificación , Quimiocina CXCL13/genética , Quimiocina CXCL13/aislamiento & purificación , Clonación Molecular , Escherichia coli , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/aislamiento & purificación , Humanos , Cuerpos de Inclusión/metabolismo , Interleucina-17/genética , Interleucina-17/aislamiento & purificación , Renaturación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sustancias Reductoras/química , Sustancias Reductoras/metabolismoRESUMEN
Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (K i < 10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C19 > 1 µM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development.