| Author/Editor | Svetina, Monika; Kraševac, Nada; Gaberc-Porekar, Vladka; Komel, Radovan | |
| Title | Improved strategy for production and purification of the human tumor necrosis factor alpha from inclusion bodies | |
| Type | članek | |
| Source | Food Technol Biotechnol | |
| Vol. and No. | Letnik 35, št. 1 | |
| Publication year | 1997 | |
| Volume | str. 23-8 | |
| Language | eng | |
| Abstract | Production of human tumor necrosis factor alpha (hTNF-alpha) 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-alpha. Electroelution from semipreparative SDS-PAGE yielded a relatively small amount of the fusion protein, while cation exchange chromatography turned out to be a conveninet method for purification of large amounts of hTNF-alpha protein. Two methods for the clevage 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 clevage by the factor Xa was only partial. We succeded in increasing the efficiency of this clevage by introducing the collagen linker in front of the factor Xa clevage site. It was shown tht thus obtained fusion protein ADK-CL-Xa-TNF was advantagenous relative to the fusion protein ADK-Xa-TNF also because its degree of precipitation during the dialysis against buffer solution, used for cleavage with factor Xa, was smaller. After the cleavage by CNBr, hTNF-alpha was isolated by electroelution from the semipreparative gel, whereas after the cleavage by the factor Xa, the cation exchange colummn 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-alpha was lower, however, the percentage of active protein was higher. | |
| Descriptors | TUMOR NECROSIS FACTOR ESCHERICHIA COLI INCLUSION BODIES TRANSFORMATION, GENETIC RECOMBINANT FUSION PROTEINS |