Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1998-09-30
2000-05-16
Brusca, John S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435455, 435325, 4353201, 536 231, 536 241, C12P 2100, C12N 1512, C12N 1563
Patent
active
060635983
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a strong homologous promoter from hamsters, nucleic acids which contain promoter and/or regulatory sequences of the ubiquitin-S27a gene, and processes for preparing heterologous gene products using such nucleic acids.
In the economically important production of proteins by the expression of recombinant genes in eukaryotic host cells, such as CHO cells, heterologous expression systems have hitherto been used, i.e. for example the promoter/enhancer and termination elements are of viral origin. The use of non-viral promoters instead of viral sequences in expression systems would be advantageous in terms of reconciling the public to genetic engineering and biotechnology and would also help to ensure the biosafety of the vector systems used for expressing genes in animal cell cultures.
Ubiquitin is a highly conserved polypeptide of 76 amino acids which can be found in large numbers in all eukaryotic cells and is coded by a diverse gene family (Reviews: Jentsch et al., 1991; Schlesinger & Bond, 1987). By modifying target proteins, ubiquitin plays a decisive role in a variety of biological processes such as the ATP-dependent protein degradation by the ubiquitin-proteosome pathway (Ciechanover, 1994). On the basis of their structure, ubiquitin genes can be divided into two groups. The first group includes the polyubiquitin genes in which ubiquitin coding units having 228 bp (=76 amino acids) are lined up in a head to tail arrangement (Jentsch et al., 1991; Schlesinger & Bond, 1987). The number of units varies from species to species, although most organisms contain two polyubiquitin genes of different lengths (Fornace et al., 1989; Wiborg et al., 1985). The promoter regions of these genes contain a TATA box and promoter/enhancer elements for a heat shock inducer (Schlesinger & Bond, 1987).
The ubiquitin fusion genes belong to the second group. These are fusions between a ubiquitin unit and a ribosomal protein (Jentsch et al., 1991; Schlesinger & Bond, 1987). The two known ubiquitin fusion genes can be identified on the basis of the differences in length and sequence of the ribosomal protein. In one case, the ribosomal protein is that of the large ribosome subunit with a length of 52 amino acids (Baker et al., 1991) whilst in the other case the ribosomal protein is that of the small ribosome subunit (designated protein S27a in mammals) with a species-dependent length of 76 to 81 amino acids (Redman & Rechsteiner, 1989). The ubiquitin part of these fusion proteins apparently supports the efficient integration of the ribosomal proteins into the ribosome (Finley et al., 1989).
The individual ubiquitin genes are expressed to different degrees in all kinds of tissues in a living organism and in various stages of development. Thus, the polyubiquitin genes are constitutively expressed at low levels which are only increased sharply under stress (Fornace et al., 1989; Schlesinger & Bond, 1987). The ubiquitin fusion genes are primarily expressed more strongly during the exponential growth phase. In terminally differentiated and growth-arrested cells, on the other hand, expression is reduced (Schlesinger & Bond, 1987; Shimbara et al., 1993; Wong et al., 1993).
The objective which the present invention sets out to achieve was to prepare strong non-viral promoters for processes for the preparation of heterologous gene products in culture cells, particularly hamster cells.
Surprisingly, a promoter of a gene was found which has an activity equivalent to the viral SV40-promoter particularly in CHO cells. This gene codes for the ubiquitin fusion protein Ub/S27a. The present invention relates to the Ub/S27a-promoter, particularly the Ub/S27a-promoter from hamsters. The invention further relates to regulator sequences in the 5' untranslated region of the Ub/S27a-gene.
The present invention also relates to a nucleic acid molecule which contains promoter sequences and/or other regulatory sequences of the Ub/S27a-gene. Preferably, the promoter sequences and/or other regulatory sequences are derived from the Ub/2
REFERENCES:
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Bergemann Klaus
Enenkel Barbara
Gannon Frank
Noe Wolfgang
Brusca John S.
Dr. Karl Thomae GmbH
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