title: Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein.
abstract: To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.
authors.affiliation: TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
authors.forename: M P
authors.initials: MP
authors.lastname: Broekhuijsen
authors.forename: I E
authors.initials: IE
authors.lastname: Mattern
authors.forename: R
authors.initials: R
authors.lastname: Contreras
authors.forename: J R
authors.initials: JR
authors.lastname: Kinghorn
authors.forename: C A
authors.initials: CA
authors.lastname: van den Hondel
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*; D014170:Transformation, Genetic; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000520:alpha-Glucosidases; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae