Expression systems

Details Expression systems Expression systems

1998-06-22

2000-12-26

Guzo, David

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 691, 4353201, 435325, 435455, 435456, 435371, 435366, 435354, 4353721, 536 231, 536 241, C12Q 168, C12N 510, C12N 1586, C12N 1563

Patent

active

061657158

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to new expressions systems, and in particular to expression systems in which a gene of interest is expressed at an optimal level. Particular examples of such expression systems are retroviral packaging cell lines and a number of preferred cell lines have been identified.
The ability of eukaryotic and prokaryotic ribosomes to reinitiate translation at an internal start codon within an mRNA sequence has previously been recognised. Studies have been reported in which the efficiency of the process, which is generally regarded as being low, has been connected with the length of the intercistronic sequence (Kozak (1987) Mol. Cell Biol. 7, 3438-3445). Selection of this sequence or spacer as 70 bp in length, and containing no other start codons, has been previously reported as being optimal for reinitiation in a eukaryotic cell line (Cosset F-L., Virology (1991) 185, 862).
The applicants have found a way in which the inefficiency associated with the translation reinitiation process can be used to good effect.
According to the present invention there is provided a recombinant expression vector comprising a gene of interest and a selectable marker gene, wherein the selectable marker gene is arranged downstream of the gene of interest and a stop codon associated with the gene of interest is spaced from a start codon of said selectable marker gene at a distance which is sufficient to ensure that translation re-initiation is required before said selectable marker protein is expressed from the corresponding mRNA.
The invention further provides a process for producing cell lines in which a gene of interest is expressed, which process comprises transforming host cells with an expression vector comprising said gene of interest and a selectable marker gene, wherein the selectable marker gene is arranged downstream of the gene of interest and a stop codon associated with the gene of interest is spaced from a start codon of said selectable marker gene at a distance which is sufficient to ensure that translation re-initiation is required before said selectable marker protein is expressed from the corresponding mRNA, and selecting those cells where expression of the selectable marker gene may be detected.
Since re-initiation of translation is a relatively inefficient process, this means that the selectable marker protein will be expressed at lower levels than the product of the gene of interest. When the marker protein is expressed at detectable levels, the gene of interest will be expressed at higher levels. This will ensure that during the subsequent selection procedure, only those cell clones which express the gene of interest at higher or optimal levels will survive. Low expressing clones will be eliminated by the selection process.
Cells transformed with the above-described expression vectors form a further aspect of the invention.
The host cells are suitably eukaryotic or prokaryotic host cells, preferably eukaryotic host cells.
The number of nucleotides in the space between the stop codon of the gene of interest and the start codon of the selectable marker will suitably be in the range of from 20-200 nucleotides, preferably from 60-80 nucleotides, even more preferably 70-80 nucleotides.
The vectors used in the process of the invention may be any of the known types, for example expression plasmids or viral vectors.
Selected cells may be cultured and if required, the protein product of the gene of interest isolated from the culture using conventional techniques. Alternatively, expression of the gene of interest may result in other desired effects, for example, where the gene of interest is included as part of a viral packaging construct.
Some experimental and clinical gene transfer protocols require the design of gene transfer vectors suitable for in vivo gene delivery (Miller, A.D. 1992. Nature 357: 455-460). Retroviral vectors are attractive candidates for such applications, because they can provide stable gene transfer and expression (Samarut J. et al., Meth. Enzymol. in press) and because

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