Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-04-24
1997-09-09
Ulm, John
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352523, 4353201, 536 234, C12N 1518, C12N 121, C12N 510
Patent
active
056655673
DESCRIPTION:
BRIEF SUMMARY
The invention relates to the recombinant preparation of PDGF-AB (rPDGF-AB), in mammalian cells as host cells, which is essentially free of the homodimeric contaminating products PDGF-AA and PDGF-BB.
It has been possible for many years to prepare individual proteins, whose genes were isolated by cloning, in different prokaryotic and eukaryotic cells, following manipulation and gene transfer. Correct folding and processing, and, where appropriate, post-translational modification as well, which are often not carried out correctly in prokaryotic and lower eukaryotic expression systems, are necessary for achieving the complete biological activity of many proteins. For this reason, mammalian cells are frequently used as hosts. In addition to this, mammalian cells are able to secrete large quantities of proteins.
For various reasons, the simultaneous preparation of two or more protein chains is often required. For example, many natural proteins are, in their functional form, composed of several subunits (e.g. antibodies). In nature, the association of the different subunits of complex proteins takes place after protein synthesis. Other components of the cellular apparatus frequently participate in this association as catalysts or controlling elements, with folding of the original structures taking place on occasion. Disturbances of the association, e.g. by an equal synthesis of the individual components, can have negative consequences both for the proteins which are to be formed and for the host cell. In nature, this system is subject to sophisticated regulation, which is for the most part cell-specific. Since this regulation is in general not adjustable in genetically manipulated cells, the alternatives explained below were developed and used for the simultaneous preparation of several foreign proteins:
1) The genes are integrated separately into expression vectors and then cotransferred in an appropriate ratio into the cells. This presupposes that several plasmid copies are taken up at the same time in a stable manner and continue to be harboured during division. The ratio of the expression of the different genes to each other depends both on the copy number and on the site of integration in the genome of the host cell. It is possible, by elaborate screening processes, to isolate cell clones which express the individual gene products in the desired ratio.
2) In order to level out the copy number, the different genes are placed in independent transcription units on one vector. While this, to a large extent, ensures stoichiometric representation of the genes, this process is also subject to problems. Thus, even if expression units having promoters of equal strength are used, it is in no way guaranteed that the mRNAs, which encode the different proteins, have the same stability and translation efficiency. Nor does the transcriptional efficiency of the two genes necessarily need to be identical. In this case, the stoichiometry of expression is produced step-wise using recombinant DNA stratagems (positioning of the transcription units with respect to each other and modulation of the strength of the individual promoters by removing or adding individual elements).
3) Bicistronic or multicistronic vectors were developed in order to avoid the problems connected with the stability of the mRNA of different transcripts. For this purpose, the individual reading frames of the gene segments--cistrons--encoding the protein chains lie on the transcription unit (expression unit). Expression of the multicistronic gene is effected using a single promoter. While the first cistron in such vectors is normally translated very efficiently, translation of the subsequent cistrons depends on the intercistronic sequences. If normal 5' untranslated sequences (5'UTR) from monocistronic genes are used for these intercistronic sequences, expession of the subsequent cistron is usually very low (as a rule, about 0.5 to 2% of the translation of the first cistron, Kaufman et al., 1987; Boel et al., 1987). It was initially possible to increase this
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Achterberg Volker
Dirks Wilhem
Dorschner Albrecht
Eichner Wolfram
Hauser Hansjorg
Beiersdorf AG
Gesellschaft fur Biotechnologische Forschung mbH
Saoud Christine
Ulm John
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