Prethodno priopćenje
In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger
Nada Kraševec
; National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
Monika Svetina
; National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
Vladka Gaberc-Porekar
; National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
Viktor Menart
; National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
Radovan Komel
; National Institute of Chemistry, POB 660, SI-1001 Ljubljana, Slovenia
Sažetak
The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis.
Ključne riječi
filamentous fungi; Aspergillus niger; heterologous protein secretion; human tumor necrosis factor α (TNFα) KEX2 cleavage site; enterokinase cleavage site; His-tag
Hrčak ID:
125298
URI
Datum izdavanja:
15.12.2003.
Posjeta: 764 *