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1.
Protein Expr Purif ; 87(2): 100-10, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23147206

RESUMO

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20-30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.


Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Quimotripsina/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Subunidades Proteicas , Tripsina/metabolismo
2.
Protein Expr Purif ; 78(1): 61-8, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21354312

RESUMO

Transient gene expression (TGE) is a well-established enabling technology for rapid generation of recombinant proteins, with Human Embryonic Kidney (HEK) and Chinese Hamster Ovary (CHO) cell lines and polyethyleneimine (PEI) as the transfection reagent being its most popular components. However, despite considerable progress made in the field, volumetric titers can still be a limiting factor causing the manipulation of increasing quantities of culture media and DNA. Here, we report a systematic analysis of TGE conditions and their influence on yields and protein quality. Guided by Design of Experiments (DoE), we conclude that TGE yields with one test antibody can be maximized by a parallel increase of cell density - 2.4 to 3.0 × 10(6)cells/mL - and PEI concentration - 24 to 30 mg/L - while maintaining a 1:1 ratio of heavy chain and light chain encoding plasmids. Interestingly, we also show that in these conditions, DNA concentration can be maintained in the 1mg/L range, thereby limiting the need for large DNA preparations. Our optimized settings for PEI-mediated TGE in HEK and CHO cells evaluated on several proteins are generally applicable to recombinant antibodies and proteins.


Assuntos
Proteínas Recombinantes/biossíntese , Transfecção/métodos , Animais , Anticorpos/genética , Anticorpos/metabolismo , Células CHO , Contagem de Células , Sobrevivência Celular/fisiologia , Clonagem Molecular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Polietilenoimina , Proteínas Recombinantes/genética , Projetos de Pesquisa
3.
Protein Expr Purif ; 75(2): 192-203, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20851186

RESUMO

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/metabolismo
4.
Proteomics ; 10(24): 4342-51, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21136589

RESUMO

CXCL12α has been shown to be selectively processed at the N- and C-termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF-MS-based method was developed. Anti-CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI-TOF-MS. The protein was found to be processed both at the C- and N-termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N-terminus, where altogether seven amino acids were removed. At the C-terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full-length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6-8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC-CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N-terminal methionine was protected against N-terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full-length CXCL12α.


Assuntos
Quimiocina CXCL12/sangue , Animais , Quimiocina CXCL12/administração & dosagem , Feminino , Humanos , Injeções Intravenosas , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL
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