RESUMO
Stromal cellderived factor1 (SDF1) is a chemokine involved in neuronal differentiation, as well as proliferation and migration. In the present study, the effects of recombinant SDF1 on neurite outgrowth for nerve regeneration and engineering were evaluated in PC12 cells. The effects of purified SDF1 protein on cell toxicity, proliferation and migration were also assessed. SDF1 significantly augmented cell proliferation in a dosedependent manner, with low cytotoxicity in PC12 cells. Cell migration also increased in the presence of SDF1. SDF1 significantly increased neurite number and length, compared with the control (untreated cells). Neurofilament mRNA levels, which are involved in neuronal differentiation, were also significantly upregulated in the presence of SDF1. These results suggested that SDF1 might prove useful for tissue engineering through induction of neuronal differentiation.
Assuntos
Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/fisiologia , Neuritos/metabolismo , Crescimento Neuronal/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Quimiocina CXCL12/isolamento & purificação , Filamentos Intermediários/genética , Microscopia Confocal , Microscopia Eletrônica de Varredura , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Cicatrização/fisiologiaRESUMO
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.
Assuntos
Anafilatoxinas/química , Anafilatoxinas/metabolismo , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/metabolismo , Ensaios de Triagem em Larga Escala , Corpos de Inclusão/química , Interleucina-17/química , Interleucina-17/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Projetos de Pesquisa , Anafilatoxinas/genética , Anafilatoxinas/isolamento & purificação , Bioensaio , Quimiocina CXCL12/genética , Quimiocina CXCL12/isolamento & purificação , Quimiocina CXCL13/genética , Quimiocina CXCL13/isolamento & purificação , Clonagem Molecular , Escherichia coli , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/isolamento & purificação , Humanos , Corpos de Inclusão/metabolismo , Interleucina-17/genética , Interleucina-17/isolamento & purificação , Renaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Substâncias Redutoras/química , Substâncias Redutoras/metabolismoRESUMO
The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1alpha (stromal cell-derived factor-1alpha) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1alpha fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP-RANTES and MBP-SDF-1alpha was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1alpha.
