The expression-secretion system of the GRAS filamentous fungus A. niger has proven to be capable of producing heterologous proteins at commercially interesting levels, however, it has also to be taken into account that it has not yet been applied for a wide range of heterologous protein products. Human tumor necrosis factor alpha (hTNF-alpha) monomer is a 17 kDa non-glycosylated protein, containing a single infra-molecular disulfide bond in its structure. In solution it exists as a compact, bell-shaped homotrimer, which is considered to be a biologically active form of this important cytokine with a wide range of biological activities. For heterologous expression of hTNF-alpha in A. niger the same strategies were used, which were proved to be the most successful for several other non-fungal proteins: a) gene-fusion with the A. niger glucoamylase GII form as a carrier-gene, with KEX2-like in vivo cleavage-site in between the genes; b) strong fungal transcription control regions and efficient secretion signals of the A. niger glucoamylase gene; and c) selection of high copy transformants due to the amdS selection marker in the protease-deficient host-strain A. niger AB1.13. No processed hTNF-alpha detected in the media or even in the cell, although specific mRNA of the expected size was observed. We could detect hTNF-alpha in the form of glucoamylase-fusion protein in the cell, regardless of the presence or absence KEX2-like in vivo cleavage-site. This glucoamylase-hTNF-alpha fusion-protein was N-glycosylated due to the glucoamylase GII part (O-glycosylation was not checked), mostly attached to the membranes, but not to the outside of the cell wall. Only 12% of hTNF-alpha added into maltodextrine production medium was proteoliticaly degraded after 24 hours, so degradation in the protease-deficient host-strain AB 1.13 compared to N402 strain was not a problem, hTNF-alpha analogue LK 802 (Cys95/148, His107/108) was also expressed with carrier-gene strategy. Cys95/148 mutation is introducing an intermolecular disulfide bond so that a more compact and stable trimeric molecule is produced, and His 107/108 mutation is introducing six histidine residues at the apical site of the molecule, providing an efficient separation method on metal affinity chromatography. However, the glucoamylase-hTNF-alpha analogue fusion-protein with both above mentioned mutations could not be successfully separated from the cell extract. In all these experiments hTNF-alpha synthetic gene optimised for E. coli bias was used, but when the gene was exchanged for natural human TNF-alpha cDNA, processed hTNF-alpha was successfully secreted into the medium. After the first screening of small amounts of transformants the yield was estimated to 13 mg/1 culture.
Fungal Genet. Newsl. 46 (Supl):
Full conference title:
Fungal Genetics Conference 20th
- Fungal Genetics Conference 20th (1999)