Open AccessCharacterization of the Kexin-Like Maturase of Aspergillus niger
Author(s) -
Ruud Jalving,
Peter J. I. van de Vondervoort,
Jaap Visser,
Peter J. Schaap
Publication year - 2000
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.66.1.363-368.2000
Subject(s) - fusion protein , aspergillus niger , biology , biochemistry , cleavage (geology) , kexin , secretion , proprotein convertase , gene , chemistry , recombinant dna , lipoprotein , paleontology , ldl receptor , cholesterol , fracture (geology)
Secreted yields of foreign proteins may be enhanced in filamentous fungi through the use of translational fusions in which the target protein is fused to an endogenous secreted carrier protein. The fused proteins are usually separated in vivo by cleavage of an engineered Kex2 endoprotease recognition site at the fusion junction. We have cloned the kexin-encoding gene ofAspergillus niger (kexB ). We constructed strains that either overexpressed KexB or lacked a functionalkexB gene. Kexin-specific activity doubled in membrane-protein fractions of the strain overexpressing KexB. In contrast, no kexin-specific activity was detected in the similar protein fractions of thekexB disruptant. Expression in this loss-of-function strain of a glucoamylase human interleukin-6 fusion protein with an engineered Kex2 dibasic cleavage site at the fusion junction resulted in secretion of unprocessed fusion protein. The results show that KexB is the endoproteolytic proprotein processing enzyme responsible for the processing of (engineered) dibasic cleavage sites in target proteins that are transported through the secretion pathway ofA. niger .