RECOVERY OF RECOMBINANT HUMAN TUMOR NECROSIS FACTOR BETA (RHTNF-B) EXPRESSED IN ESCHERICHIA COLI AS INCLUSION BODIES

Abstract

Present study deals with the purification and renaturation of recombinant human tumor necrosis factor beta (rhTNF-?) from inclusion bodies (IBs). Recombinant E.coli K12 B101 strain cloned with rhTNF-? gene was employed in this study. A procedure for purification and renaturation of rhTNF-? from inclusion bodies has been designed. The procedure includes inclusion bodies washing with specific wash buffer (Triton X-100/ EDTA / lysozyme / PMSF), inclusion bodies solubilization with 8 M alkaline urea, purification with CM-Sepharose and DEAE-Sepharose ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with ultrafiltration membrane. The conditions for IBs recovery, IBs solubilization and rhTNF-? purification have been optimized. For IBs recovery, after cell lysis, the centrifugation condition of 12000g for 10 minutes is employed to ensure more than 90 % of IBs be recovered. Subsequently, wash buffer containing a nonionic detergent (Triton X-100), a chelating agent (EDTA), a protease inhibitor (PMSF) and a cell lytic enzyme (lysozyme) was used to extract aqueous-insoluble impurities from IBs and remain IBs in insoluble form. IBs solubilization can be achieved by incubation of IBs in buffer containing 8 M urea at pH 10.5 for at least 90 minutes. Denatured rhTNF-? can be purified by ion-exchange chromatography in the presence of 6 M of urea at pH 7 .5. The procedure and operating conditions have been verified experimentally at laboratory scale with a product purity of more than 90 percent and over all recovery of about 30 percent. The characteristics of the renatured protein were identical with those purified from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929, (3) N-terminal amino acid sequence, and (4) gel filtration.