Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-02-20
1998-07-21
Guzo, David
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525411, 4352542, 43525421, 43525422, 43525423, 4352568, 4353201, 530344, 530350, 530363, 530364, C12P 2102, C12N 119, C07K 114
Patent
active
057834237
DESCRIPTION:
BRIEF SUMMARY
1. Field of the Invention
The present invention relates to the production of heterologous proteins by yeast species and more particularly to an adaptation of the yeast in which the protein is produced.
2. Background and Prior Art
In recent years, yeasts have been widely used as host organisms for the production of heterologous proteins (reviewed by Romanos et al, 1992), including recombinant human albumin (rHA) (Sleep et al, 1990, 1991; Fleer et al, 1991). Yeasts are readily amenable to genetic manipulation, can be grown to high cell density on simple media, and as eukaryotes are suitable for the production of secreted as well as cytosolic proteins.
When yeasts are utilised to produce a desired heterologous protein by secretion into the growth medium, a large number of host-derived proteins may also be present, including other proteins secreted by the host but also intracellular proteins present in the supernatant as the result of leakage from cells or cell lysis. In processes in which the protein is not secreted, there is of course an even higher level of contamination with intracellular yeast proteins. It is necessary to purify the desired protein and to remove these contaminating proteins from the preparation; such methods have been disclosed in WO 92/04367 and EP 524 681. The majority of contaminating proteins will have physicochemical properties sufficiently different from the desired protein to permit efficient separation by standard techniques, such as ion exchange or size exclusion chromatography. The prior art gives the impression that such proteins can be satisfactorily removed by such techniques; see, for example EP 524 681 (Gist-brocades), EP 570 916 (Green Cross) and EP 464 590 (Green Cross). Indeed, we have developed sophisticated chromatographic techniques (unpublished) to remove contaminating proteins from desired proteins.
SUMMARY OF THE INVENTION
We have now also adopted a different approach and have identified the gene responsible for a protein, namely the HSP150 gene, which co-purifies with recombinant human albumin (rHA) and, in principle, with other desired proteins. In accordance with the invention, we eliminate the contaminating protein from the initial fermentation, rather than develop highly sophisticated and complex means of removal during purification. This protein was not previously known to be a co-purifying contaminant.
In one aspect of the invention, the HSP150 gene is functionally deleted from the genome of the host. This has not caused any detrimental effects on production of the desired protein and removes a potential contaminant that has proven difficult to remove by standard purification techniques. Despite the presence of at least two closely related genes encoding proteins very similar to Hsp150, PIR1 and PIR3, in such modified yeast, rHA purified from these organisms does not contain detectable levels of any protein from this family.
The S. cerevisiae Hsp150 protein was originally described by Russo et al (1992) and was shown to be produced constitutively, to be extensively O-glycosylated and to be secreted efficiently into the growth medium. A 7-fold increase in the level of Hsp150 protein was seen when cells grown at 28.degree. C. were shifted to 37.degree. C. Makarow has proposed preparing fusions of Hsp150 (or fragments thereof) and a desired protein, in order to achieve enhanced, controllable secretion (WO 93/18167). The HSP150 gene encodes a primary translation product of 413 amino acids, including an N-terminal secretion signal sequence of 18 amino acids that is not present in the mature protein. A further post-translational processing event occurs C-terminal to a pair of basic residues to yield two subunits of 54 and 341 amino acids which remain associated. The 341 amino acid subunit contains 11 tandem repeats of a 19 amino acid sequence, the function of which is unknown. Homologues of the HSP150 gene were found in Torulaspora delbrueckii, Kluyveromyces marxianus and Schizosaccharomyces pombe (Russo et al, 1992).
The same protein has been designated the PI
REFERENCES:
Mol Gen Genet, vol. 239, pp. 273-280, 1993.
Bio/Technology, vol. 8, pp. 42-46, 1990.
Quirk Alan Victor
Wood Patricia Carolyn
Biswas Naomi S.
Delta Biotechnology Ltd.
Guzo David
Newman Irving
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