Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-03
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S372300, C435S320100, C435S455000, C435S377000, C435S369000, C435S358000
Reexamination Certificate
active
06653101
ABSTRACT:
The present invention relates to a method for producing proteins by recombinant DNA technology and to permanently transfected host cells for use in the method.
In recent years, recombinant DNA technology has advanced to the stage where, in general, it is readily possible to prepare a gene, that is a DNA sequence which encodes a desired protein. The gene may encode only a desired protein product or may encode a pro- or prepro-protein which, after translation, is cleaved to produce the desired final product. The gene may be prepared, for instance, (1) by isolating messenger RNA (mRNA) and using this as a template for the production of complementary DNA (cDNA) by reverse transcription, (2) by isolating the natural gene from genomic DNA using appropriate probes and restriction enzymes, (3) by synthesizing the gene from its component nucleotides or (4) by using a combination of these techniques.
It is also well known that a prepared gene can be placed in a vector, such as a plasmid or phage vector, under the control of appropriate 5′ and 3′ flanking sequences which allow the gene to be transcribed into mRNA and then translated into protein. Many important 5′ and 3′ flanking sequences, such as translation start and stop codons, TATA boxes, promoters, enhancers and polyadenylation sites, have been identified. The part of a vector including the gene and the 5′ and 3′ sequences is herein referred to as a transcription unit.
In general, the skilled person will be able readily to construct an expression vector and use it to transfect or transform a host cell such that the host cell is able to produce the desired final protein product. A variety of host cells can be used. Early work was carried out using prokaryotic microorganisms such as
E. Coli
. These hosts had the disadvantages that they do not have the necessary mechanisms to cleave efficiently pro- or prepro- sequences from proteins, they are unable to glycosylate proteins, they cannot cope with genes containing introns, and they generally do not secrete the proteins when formed.
There has therefore been a tendency towards the use of eukaryotic host cells. These generally avoid most of the disadvantages of prokaryotic host cells. However, eukaryotic host cells generally have more stringent requirements for culturing and also have slower growth rates. It is therefore not readily possible to produce large quantities of a product merely by culturing a eukaryotic host cell transformed with an expression vector.
There has therefore been considerable effort expended on increasing the amount of product which can be produced by a single eukaryotic host cell. Two of the main factors which control the amounts of product which a host cell can produce are gene copy number and the efficiency of transcription of each gene copy. There have been a number of proposals for increasing host cell productivity either by increasing gene copy number or by increasing transcription efficiency for each gene copy.
The most common method used to increase gene copy number is selection for gene amplification. In gene amplification, for instance as described in EP-A-0 045 809 or U.S. Pat. No. 4,634,665, a host cell is transformed with linked or unlinked genes. The first gene encodes a desired protein and the second gene encodes a selectable marker, such as DHFR. Cell lines containing both genes are then cultured in ever increasing concentrations of a toxic agent, the effect of which is nullified by the product of the selectable marker gene. It has been found that those cell lines which survive in the higher concentrations of the toxic agent have an increased copy number of both the selectable marker gene and the desired product gene. Thus, the host cell having the amplified number of gene copies can produce a larger amount of the desired protein than the original cell lines.
A disadvantage of gene amplification is that it is a laborious task to produce a highly productive cell line. Moreover, once the cell line has been produced, it is frequently necessary to retain it in a culture medium containing a high level of the selective agent. If this is not done, the selective pressure is released and the excess copies of the genes for the selectable marker and the desired protein may be eliminated. Using a culture medium containing a toxic agent causes problems in the purification of the desired product.
Another approach to increasing productivity is to increase the efficiency of transcription for each gene copy. This can be achieved by selection of a promoter which can be switched on by a stimulus, such as a toxic agent or heat. Alternatively, the gene for the desired product may merely be put under the control of a strong promoter. Although these approaches have to a certain extent been successful, they have not totally succeeded in raising productivity to commercially desirable levels.
There is therefore a need for a method for increasing the productivity of eukaryotic host cells containing genes encoding desired proteins.
It has been known for some long time that there are viral genes which, when translated, produce activator proteins which cause the activation of other genes in the viral genome. The activator proteins may act directly on recognition sites upstream of the viral promoter/enhancer sequence. Alternatively, the activator protein may interact with one or more other proteins which act on the recognition site to increase transcription. This activation of genes by an activator protein is often called transactivation.
It has also been found in a number of cases that an activating protein produced by one virus can act on recognition sites in other viruses or on certain cellular gene promoters to cause transactivation of the genes associated with the activation site.
References [
1
] to [
30
] listed in the attached bibliography are examples of the work done on transactivation, with particular reference to the Adenovirus E1A proteins.
The E1A region of adenoviruses encodes two major early proteins of 289 and 243 amino acids respectively. These are produced from two differently spliced mRNAs of 13S and 12S respectively. These E1A proteins are multifunctional and have been shown to play an essential role in cellular immortalization and transformation. The E1A gene product is also known to regulate the expression of certain viral and cellular genes in a positive or negative manner.
The 289 and 243 proteins are identical except in that the 243 protein lacks a 46 amino acid region towards the centre of the 289 protein. The 289 protein has been investigated and several functions have been assigned to certain domains thereof. The 289 protein is shown in
FIG. 1
to which reference is now made. Domain 1 mediates induction of DNA synthesis. Domain 2 mediates induction of mitosis, cellular immortalisation and transformation, and transcription repression. Domain 3 mediates indirect transactivation of viral and cellular genes. Domain 4 mediates nuclear localisation.
The mechanism for gene repression and cellular transformation is unclear, but point mutations within Domain 2 are able to abolish both repression and transformation without affecting the other functions.
WO-A-89/05862 (Invitron) describes the use of E1A to immortalise or extend the life of cells from primary cultures which would otherwise senesce after a limited number of cell generations in culture. In particular, the application describes the use of E1A to extend the lifespan of the human colon mucosa cells CCD 18 Co to allow continuous secretion of the tPA produced natively from these cells. This approach exploits the known immortalising—oncogene function of the 13S and 12S mRNAs from the E1A gene. It does not rely on transactivation.
More is known about the mechanism of transactivation, but even now the full mechanism has not been elucidated. It is known that the E1A 289 protein is phosphorylated and that somehow the active E1A protein induces phosphorylation of various DNA binding transcription factors, such as TFIId, ATF and TFIIIc, thus increasin
Bebbington Christopher Robert
Cockett Mark Ian
Yarranton Geoffrey Thomas
Alusuisse Holdings A.G.
Ketter James
Marvich M
Nixon & Vanderhye P.C.
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