Recombinant production of bioactive human TNF-alpha by SUMO-fusion system--high yields from shake-flask culture
| Autor: | Andrea Sinz, Manja Gloser, Andreas Hoffmann, Mathias Q. Müller, Rainer Rudolph, Sven Pfeifer |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
SUMO-1 Protein
medicine.medical_treatment Recombinant Fusion Proteins Cell Culture Techniques Cleavage (embryo) medicine.disease_cause Chromatography Affinity law.invention Mice law Cell Line Tumor medicine Escherichia coli Animals Humans Chromatography High Pressure Liquid Protease Chromatography Cell Death Chemistry Tumor Necrosis Factor-alpha Circular Dichroism Biological activity Biochemistry Solubility Cell culture Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA Chromatography Gel Tumor necrosis factor alpha Electrophoresis Polyacrylamide Gel Biotechnology |
| Zdroj: | Protein expression and purification. 72(2) |
| ISSN: | 1096-0279 |
| Popis: | Tumor necrosis factor (TNF-alpha) inhibitors, used for the treatment of common inflammatory diseases, currently belong among the most important biotechnologically produced pharmaceuticals. So far four TNF-alpha antagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerous approaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areas in the treatment of TNF-alpha -related diseases. Both the fundamental understanding of disease-related TNF-alpha activity and the subsequent development of corresponding drug candidates demand the availability of large amounts of TNF-alpha as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-alpha in Escherichia coli shake-flask cultures and the subsequent purification of the mature protein. Using the advantages of SUMO-fusion technology we were able to produce protein with an authentic N-terminus in high yield. Two immobilized metal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size-exclusion chromatography were utilized to purify the protein. The protein was obtained from the last chromatography step as a trimer, while purity was at least 96% as estimated by SDS-PAGE. The identity of the protein was confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-alpha was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect