ALL libraries (COBIB.SI union bibliographic/catalogue database)
  • In vivo and in vitro cleavage of glucoamylase-TNF[alpha] fusion protein secreted from Aspergillus niger
    Kraševec, Nada ...
    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 alpha (TNFalpha) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFalpha, 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 TNF alpha 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.
    Source: Food technology and biotechnology : journal of the Faculty of Food Technology and Biotechnology University of Zagreb. - ISSN 1330-9862 (Vol. 41, no. 4, 2003, str. 345-351)
    Type of material - article, component part
    Publish date - 2003
    Language - english
    COBISS.SI-ID - 2958618

Shelf entry