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

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.

Intracellular protein production in Trichoderma reesei (Hypocrea jecorina) with hydrophobin fusion technology.

Insufficient accumulation and the lack of efficient purification methods are the two major bottlenecks hindering the recombinant production of many proteins. Alternative production schemes are urgently needed for proteins that remain challenging to express and purify with conventional techniques. We have found that hydrophobin fusions targeted to endoplasmic reticulum (ER) can enhance the expression of target proteins simultaneously providing means for straightforward purification. Here we show that hydrophobin fusion technology induces formation of large protein bodies in the filamentous fungus Trichoderma reesei. The fusion protein remained soluble in the ER-derived protein bodies. A simple and scalable aqueous two-phase system was demonstrated to purify the hydrophobin fusion protein GFP-HFBI from the complex intracellular extracts with a recovery of up to 62%.

Expression of the Ciona intestinalis alternative oxidase (AOX) in Drosophila complements defects in mitochondrial oxidative phosphorylation.

Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinson's disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.