Recombinant production of bioactive human TNF-alpha by SUMO-fusion system--high yields from shake-flask culture.
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.
Hoffmann
Andreas
A
NWG Künstliche Bindeproteine, Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany.
Müller
Mathias Q
MQ
Gloser
Manja
M
Sinz
Andrea
A
Rudolph
Rainer
R
Pfeifer
Sven
S
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014409:Tumor Necrosis Factor-alpha