Recombinant production of bioactive human TNF-α by SUMO-fusion system - High yields from shake-flask culture

We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-α 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-α was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay.