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, 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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 164–174, 2001.