Details
| Originalsprache | Englisch |
|---|---|
| Seiten (von - bis) | 164-174 |
| Seitenumfang | 11 |
| Fachzeitschrift | Biotechnology and bioengineering |
| Jahrgang | 76 |
| Ausgabenummer | 2 |
| Publikationsstatus | Veröffentlicht - 8 Aug. 2001 |
| Extern publiziert | Ja |
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.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biotechnologie
- Chemische Verfahrenstechnik (insg.)
- Bioengineering
- Immunologie und Mikrobiologie (insg.)
- Angewandte Mikrobiologie und Biotechnologie
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in: Biotechnology and bioengineering, Jahrgang 76, Nr. 2, 08.08.2001, S. 164-174.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Production of tissue plasminogen activator (t-PA) in Aspergillus niger
AU - Wiebe, Marilyn G.
AU - Karandikar, Atul
AU - Robson, Geoff D.
AU - Trinci, Anthony P.J.
AU - Candia, Juana L.Flores
AU - Trappe, Susanne
AU - Wallis, Gregg
AU - Rinas, Ursula
AU - Derkx, Patrick M.F.
AU - Madrid, Susan M.
AU - Sisniega, Heidi
AU - Faus, Ignacio
AU - Montijn, Roy
AU - van den Hondel, Cees A.M.J.J.
AU - Punt, Peter J.
PY - 2001/8/8
Y1 - 2001/8/8
N2 - 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.
AB - 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.
KW - Aspergillus niger
KW - Cyclophilin B
KW - Protease-deficient
KW - Tissue plasminogen activator (t-PA)
KW - Unfolded protein response (UPR)
UR - http://www.scopus.com/inward/record.url?scp=0034850166&partnerID=8YFLogxK
U2 - 10.1002/bit.1156
DO - 10.1002/bit.1156
M3 - Article
C2 - 11505386
AN - SCOPUS:0034850166
VL - 76
SP - 164
EP - 174
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
SN - 0006-3592
IS - 2
ER -