title: Production of tissue plasminogen activator (t-PA) in Aspergillus niger.
abstract: A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.
authors.affiliation: School of Biological Sciences, University of Manchester, Manchester, UK. mgw@bio.auc.dk
authors.forename: M G
authors.initials: MG
authors.lastname: Wiebe
authors.forename: A
authors.initials: A
authors.lastname: Karandikar
authors.forename: G D
authors.initials: GD
authors.lastname: Robson
authors.forename: A P
authors.initials: AP
authors.lastname: Trinci
authors.forename: J L
authors.initials: JL
authors.lastname: Candia
authors.forename: S
authors.initials: S
authors.lastname: Trappe
authors.forename: G
authors.initials: G
authors.lastname: Wallis
authors.forename: U
authors.initials: U
authors.lastname: Rinas
authors.forename: P M
authors.initials: PM
authors.lastname: Derkx
authors.forename: S M
authors.initials: SM
authors.lastname: Madrid
authors.forename: H
authors.initials: H
authors.lastname: Sisniega
authors.forename: I
authors.initials: I
authors.lastname: Faus
authors.forename: R
authors.initials: R
authors.lastname: Montijn
authors.forename: C A
authors.initials: CA
authors.lastname: van den Hondel
authors.forename: P J
authors.initials: PJ
authors.lastname: Punt
mesh_terms: D001234:Aspergillus niger / Q000235:genetics*; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D015152:Blotting, Northern; D015153:Blotting, Western; D021983:Cyclophilins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019696:Peptidylprolyl Isomerase; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: C081786:cyclophilin B; D010959:Tissue Plasminogen Activator; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D005947:Glucose