Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Patent
1995-04-18
1998-02-03
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
435 691, 4352542, 43525421, 536 232, C12N 115, C12N 116, C12P 2102, C07H 2104
Patent
active
057143773
DESCRIPTION:
BRIEF SUMMARY
This invention relates to improved fungal cells and methods for producing recombinant products of improved quality and in high yields. More specifically, the present invention relates to fungal cells carrying specific modifications within their DNA sequences which cause them to exhibit at least a reduced capacity for O-glycosylating homologous and/or heterologous proteins, and the use of these cells as host cells to produce high yields of recombinant products.
The development of recombinant DNA technology has made possible the production of foreign products in host cells in which exogenous DNA sequences coding for those products have been introduced. The advantage of this technology is that products can be produced in high yields, in highly purified form, with no risk of contamination such as viral contamination (AIDS, hepatitis B, etc.). These recombinant techniques have been widely used for the production of recombinant proteins in prokaryotic as well as al., J. Bacteriol. 169 (1987), 2917!, Streptomyces, and Corynebacterium (EP 433 117). Eukaryotic host cells include plant cells, animal cells and fungal cells.
However, the large-scale production of recombinant products by these techniques is still limited, due to problems of expression efficiency of these exogenous DNA sequences, due also to vector instability and to intracellular degradation of the recombinant products by the host cell in which they are made. Concerning expression efficiency, efforts have been made to isolate strong promoters, leading to increased expression levels of exogenous DNA sequences, and therefore to increased production levels of recombinant products. Various systems have also been developed in order to increase the stability of the vectors within the host cells, the most frequently used of which consisting in the insertion on the vector of an antibiotic resistance gene enabling recombinant host cells to survive and grow in a selective medium. With respect to intracellular degradation, several mutant cells lacking or having a reduced protease activity have been disclosed, thereby limiting the capacity of said cells to degrade recombinant products.
However, additional problems still limit large-scale production and pharmaceutical use of recombinant products. One of these arises from the fact that recombinantly produced products are often different from their natural counterparts. For example, bacterial host cells do not possess all the post-translational mechanisms required for maturation of mammalian polypeptides. Accordingly, said mammalian polypeptides produced in bacteria are often immature or not correctly refolded. Furthermore, bacterial host cells generally introduce an additional N-terminal methionine to the products.
Recombinant products produced in heterologous eukaryotic hosts also usually differ from their naturally-occurring counterpart in their glycosylation content. This may concern the presence versus absence of any carbohydrate structure, the localization of said carbohydrate structure on the product, as well as the nature of the carbohydrate. More specifically, it has been shown that yeast-derived recombinant products often bear additional unnatural O-glycans compared to their natural counterpart. For instance, it has been shown that, while human serum insulin-like growth factor I (IGF-I) is not glycosylated, its recombinant form produced in S. al., FEBS Letters 248 (1989), 111!. In the same way, it has been shown that human platelet-derived growth factor (PDGF) and human GM-CSF display Environ. Mass Spectrometry 19 (1990), 665; BIO/TECHNOLOGY 5 (1987), 831!. This abnormal O-glycosylation is the result of important differences between the glycosylation mechanisms of mammalian (human) cells and those of other eukaryotic cells, such as yeasts. In this respect, it has been observed that O-glycosylation in fungal cells (including yeasts and filamentous fungi) proceeds in a similar and unusual way so far not observed in any other organism.
The occurrence of this undesirable O-glycosylation on fungal-derived recomb
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Fleer Reinhard
Fournier Alain
Strahl-Bolsinger Sabine
Tanner Widmar
Lau Kawai
Rhone-Poulenc Rorer S.A.
Wax Robert A.
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