Improved Strategy for Production and Purification of the Human Tumor Necrosis Factor α from Inclusion Bodies

Monika Svetina, Nada Kraševac, Vladka Gaberc-Porekar, Radovan Komel

National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia

Article history:
Received January 20, 1997 
Accepted March 21 1997

Key words:
human tumor necrosis factor α, inclusion bodies, transformation and expression studies

Summary:
Production of human tumor necrosis factor α (hTNF-α) in the expression system E. coli/pMAX resulted in high expression of the 25 kDa fusion protein appearing as an insoluble fraction in inclusion bodies. Several isolation procedures, cleavage methods and renaturation procedures are described, which have been investigated in order to obtain a high amount of active hTNF-α. Electroelution from semipreparative SDS-PAGE yielded a relatively small amount of the fusion protein, while cation exchange chromatography turned out to be a convenient method for purification of large amounts of hTNF-α protein. Two methods for the cleavage of fusion protein were checked: chemical by CNBr and enzymatic by the blood coagulation factor Xa. CNBr treatment of the fusion protein ADK-Xa-TNF completely cleaved the fusion protein even at pH of 6.8, whereas cleavage by the factor Xa was only partial. We succeeded in increasing the efficiency of this cleavage by introducing the collagen linker in front of the factor Xa cleavage site. It was shown that thus obtained fusion protein ADK-CL-Xa-TNF was advantageous relative to the fusion protein ADK-Xα-TNF also because its degree of precipitation during the dialysis against buffer solution, used for cleavage with factor Xα, was smaller. After the cleavage by CNBr, hTNF-α was isolated by electroelution from the semipreparative gel, whereas after the cleavage by the factor Xα, the cation exchange column was used. In both cases dialysis was used for renaturation of the protein. The second procedure involving cation exchange chromatography turned out to be more efficient; the quantity of hTNF-α was lower, however, the percentage of active protein was higher.