Additional file 1: Table S1âS21. Lists of primers used in PCR experiments

BA3402 Protein production

Croute, Laurence

Glykos Finland Oy

Helk, Bernhard

Landowski, Christopher

Mustalahti, Eero

Novartis Pharma AG

Ostermeier, Christian

Saarinen, Juhani

Saloheimo, Markku

Sivasiddarthan, Dhinakaran

Sommer, Benjamin

Wahl, Ramon

Westerholm-Parvinen, Ann

PubMed

Background: The filamentous fungus Trichoderma reesei has tremendous capability to secrete over 100 g/L of proteins and therefore it would make an excellent host system for production of high levels of therapeutic proteins at low cost. We have developed T. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. Protease activity has been the major hindrance to achieving high production levels. We have constructed a series of interferon alpha-2b (IFNa-2b) production strains with 9 protease deletions to gain knowledge for further strain development. Results: We have identified two protease deletions that dramatically improved the production levels. Deletion of the subtilisin protease slp7 and the metalloprotease amp2 has enabled production levels of IFNa-2b up to 2.1 and 2.4 g/L, respectively. With addition of soybean trypsin protease inhibitor the level of production improved to 4.5 g/L, with an additional 1.8 g/L still bound to the secretion carrier protein. Conclusions: High levels of IFNa-2b were produced using T. reesei strains with reduced protease secretion. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage

Landowski, Christopher P.

VTT Research System

Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

Additional file 4: Figure S3. Protease activity measurements of purified fractions from culture supernatant sampled on day 4 and 5. The fractions from the pepstatin columns were measured at pH 4.5 and the SBTI fractions at pH 5.5. The protease activity on day 5 was higher than on day 4

Christopher Landowski (3521069)

Eero Mustalahti (3521063)

Ramon Wahl (244472)

Laurence Croute (3521075)

Dhinakaran Sivasiddarthan (3521066)

Ann Westerholm-Parvinen (788065)

Benjamin Sommer (3521072)

Christian Ostermeier (788070)

Bernhard Helk (255486)

Juhani Saarinen (788071)

Markku Saloheimo (62875)

FigShare

MOESM4 of Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

MOESM1 of Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

Abstract Background The filamentous fungus Trichoderma reesei has tremendous capability to secrete over 100 g/L of proteins and therefore it would make an excellent host system for production of high levels of therapeutic proteins at low cost. We have developed T. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. Protease activity has been the major hindrance to achieving high production levels. We have constructed a series of interferon alpha-2b (IFNα-2b) production strains with 9 protease deletions to gain knowledge for further strain development. Results We have identified two protease deletions that dramatically improved the production levels. Deletion of the subtilisin protease slp7 and the metalloprotease amp2 has enabled production levels of IFNα-2b up to 2.1 and 2.4 g/L, respectively. With addition of soybean trypsin protease inhibitor the level of production improved to 4.5 g/L, with an additional 1.8 g/L still bound to the secretion carrier protein. Conclusions High levels of IFNα-2b were produced using T. reesei strains with reduced protease secretion. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage

Additional file 7: Figure S6. Immunoblot detecting IFNα-2b production from bioreactor cultivations FTR108_R1 (M668 Δsep1), FTR108_R4 (M671 Δpep9), FTR108_R5 (M672 Δpep11), FTR108_R6 (M673 Δslp7), FTR108_R7 (M674 Δamp2), and FTR109_R9 (M577). Strains were grown in 20 g/L yeast extract, 40 g/L cellulose, 80 g/L cellobiose, 40 g/L sorbose, pH 4.5. The supernatants were diluted so that 0.1 µl was loaded per well. Standard amounts of IFNα-2b corresponding to 50, 100, and 200 ng were used to generate a standard curve. The full length IFNα-2b runs around 17 kDa and the carrier bound material runs at 75 kDa

MOESM7 of Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

Christopher P. Landowski

Eero Mustalahti

Ramon Wahl

Laurence Croute

Dhinakaran Sivasiddarthan

Ann Westerholm-Parvinen

Benjamin Sommer

Christian Ostermeier

Bernhard Helk

Juhani Saarinen

Markku Saloheimo

Springer - Publisher Connector

Additional file 6: Figure S5. Immunoblot detecting IFNα-2b production. A. 1 liter bioreactor cultures Triab116-118. The M669, M670, and M672 were grown in 40 g/L lactose, 20 g/L spent grain extract, 20 g/L whole spent grain, 5 g/L (NH4)2SO4, 5 g/L KH2PO4 at pH 4.5, 28 °C. The base level of the control M577 strain was 0.50 g/L. The supernatants were diluted so that 0.2 µl was loaded per well. IFNα-2b standards corresponding to 50, 100, 200 ng were loaded for quantification. B. 1 liter bioreactor cultures Triab119 and 121. The M673 and M674 strains were grown in same media. The supernatants were diluted so that 0.2 µl was loaded per well. The base level of the control M577 strain was 0.50 g/L. IFNα-2b standards corresponding to 50, 100, 200 ng were loaded for quantification

MOESM6 of Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

http://opac.ll.chiba-u.jp/da/curator/900109867/KJ00004743057.pdf