PREPARATION AND PURIFICATION OF 3-FLUORO-TYROSINE LABELED TUMOR-NECROSIS-FACTOR-ALPHA AND PRELIMINARY F-19 N.M.R. INVESTIGATION
- JK SAGOO
- LC CHAPLIN
- NRA BEELEY
- C IAONNOU
- AM NESBITT
- SJB TENDLER
Affiliations: UNIV NOTTINGHAM,DEPT PHARMACEUT SCI,BIOPHYS & SURFACE ANAL LAB,NOTTINGHAM NG7 2RD,ENGLAND. CELLTECH LTD,SLOUGH SL1 4EN,BERKS,ENGLAND.
- Published online on: October 1, 1994 https://doi.org/10.3892/ijo.5.4.937
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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.