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.

The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger

“…Approaches to overcome this problem have mainly focussed on strain improvement (Berka et al, 1990;Mattern et al, 1992;van den Hombergh et al, 1995van den Hombergh et al, , 1997bZheng et al, 1998;Moralejo et al, 2000;Wiebe et al, 2001;Moralejo et al, 2002). In addition, the use of fungal strains with growth characteristics (e.g.…”

“…Based on the above-mentioned findings, ammonium seems to be the preferred nitrogen source to repress protease activity. Excess ammonium has been suggested as a means to reduce proteolytic degradation of heterologous proteins (Wiebe et al, 2001;Wiebe, 2003). However, the effect of ammonium as the nitrogen source was less advantageous as soon as derepression of extracellular proteases occurred, for instance due to carbon source depletion.…”

Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

“…In general, A. niger is an excellent host for the production of fungal proteins but high concentrations of heterologous (non-fungal) products are notoriously difficult to obtain [2,3]. Though commercially interesting levels of recombinant bovine chymosin, an enzyme used for cheese manufacture, have already been reported in the early nineties [4,5], other proteins, for example, biopharmaceuticals such as interleukin-6 [3], tissue plasminogen activator [6], single-chain antibodies [7], and hepatitis B pseudoviral particles [8] are still just found in milligram per liter quantities in the culture medium. Unsatisfactory low yields are often caused by proteolytic degradation of the target protein [6,9], with improvements being obtained by modifying culture conditions [6,10,11] and developing strains with less proteases [12,13].…”

“…Though commercially interesting levels of recombinant bovine chymosin, an enzyme used for cheese manufacture, have already been reported in the early nineties [4,5], other proteins, for example, biopharmaceuticals such as interleukin-6 [3], tissue plasminogen activator [6], single-chain antibodies [7], and hepatitis B pseudoviral particles [8] are still just found in milligram per liter quantities in the culture medium. Unsatisfactory low yields are often caused by proteolytic degradation of the target protein [6,9], with improvements being obtained by modifying culture conditions [6,10,11] and developing strains with less proteases [12,13]. Moreover, the complex and not well understood impact of environmental conditions on fungal morphology and resulting effects on protein production and excretion imposes an additional handicap for a more widespread distribution of Aspergillus as a recombinant protein producer.…”

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

“…glycosylation and proteolytic processing. Among them are the phytase (Shah et al 2008), the xylanase (Bakri et al 2008), the glycosylated fructofuranosidase SucA (Zuccaro et al 2008), the laccase from Trametes versicolor (Bohlin et al 2006), the tissue plasminogen activator (t-PA) (Wiebe et al 2001), humanized antibodies (Ward et al 2004), human lymphotoxin α (Krasevec et al…”