Preparation and purification of 3-fluoro-tyrosine labeled tumor-necrosis-factor-alpha and preliminary f-19 N.m.R. Investigation

A one-step protocol for the purification of recombinant human tumour necrosis factor-alpha (TNF alpha) has been developed based on the use of antibody affinity chromatography. The method allows for the preparation of large amounts of the protein (>15 mg). The overall recovery of the purified material from Esherichia coli lysate after buffer exchange into 0.8% mannitol is 48%, with no apparent loss of bioactivity. This method has been utilized for the preparation of 3-fluoro-tyrosine labelled human TNF alpha. Data indicate that the protein produced in minimal media is a heterotrimer consisting of two 17 kDa monomers and one proteolytically cleaved 14 kDa unit. Preliminary F-19 n.m.r. spectroscopy indicated that the 3-fluoro-tyrosine labelled protein is suitable for further study using this technique.