3/25/2023 0 Comments Adav noti bio![]() ![]() From this production strain we made a series of protease gene deletions to find out which deletions were most beneficial to the IFNα-2b production level. In the current report we have improved the previously reported protease deletion strain by first removing the pep5 aspartic protease and then constructed an IFNα-2b production strain. In this earlier work we have deleted seven of the most problematic proteases consecutively from the same strain. We have previously reported identifying 13 major protease enzymes and making deletion strains to reduce the total secreted protease activity. reesei for use as a therapeutic protein production host with particular focus on reducing the secreted protease activity. In microbial production systems the protease problem has been reduced or overcome by deleting multiple protease genes. Production of fungal proteases has long been identified as a significant barrier to achieving high production levels of heterologous proteins. reesei is a low cost production system that can be cultivated on inexpensive medium with relatively short cultivation times. reesei was over 100 g per liter, thus it has tremendous prospects to produce large amounts of therapeutic proteins based upon its excellent secretion abilities. The highest published amount of extracellular protein produced by T. reesei have obtained the generally recognized as safe (GRAS) status by the U.S. It is suitable for large scale fermentation processes and has a long history of safe use in the enzyme production industry. The filamentous fungus Trichoderma reesei is one of the main producers of lignocellulose degrading enzymes used by enzyme industries world-wide. The maximum expression in Pichia pastoris has been reported to be around 600 mg/L. Avoiding these disadvantages, IFNα-2b has been successfully expressed in several secreted systems. The best yield of IFNα-2b after refolding and purification was reported to be 3 g/L from E. The interferon expressed in E.coli often forms insoluble, misfolded inclusion bodies that need solublization and refolding steps that could affect the integrity of the refolded proteins. The yield of recombinant interferon from E.coli is by far higher than the other systems reported, but there are several drawbacks. Several host systems have been used for production of IFNα-2b including Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, Pichia pastoris, Lactococcus lactis, Yarrowia lipolytica, and mammalian cells. It is approved around the world for the treatment of various diseases including chronic hepatitis C, chronic hepatitis B, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, follicular lymphoma, carcinoid tumor, and malignant melanoma. ![]() Thus, low cost production of IFNα-2b is necessary to provide patients with affordable therapy. IFNα-2b is on the World Health Organization’s list of essential medicines. Interferons are considered as a primary line of defence for the host immune system against infectious agents and tumor progression. Interferon alpha-2b (IFNα-2b) is an important cytokine used for antiviral and anticancer therapy. Further strain development can be done to improve the production system by reducing protease activity and improving carrier protein cleavage. reesei strains with reduced protease secretion. High levels of IFNα-2b were produced using T. 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. 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. We have identified two protease deletions that dramatically improved the 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. Protease activity has been the major hindrance to achieving high production levels. reesei strains suitable for production of therapeutic proteins by reducing the secreted protease activity. 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. ![]()
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