Production of proteins, plasmids coding therefor and organisms containing such plasmids

Plasmids that contain: (1) an origin of replication; (2) an additional sequence required for plasmid replication or preferably a receptor giving a selective advantage; and (3) two expression cassettes each of which are located between (1) and (2) but are separated from (1) and (2) from each other; and that can be liberated from inverted repeat sequences (other than in (3)) are highly persistent though successive generations of germs comprising them.


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1. Field of the Invention

THIS INVENTION relates to the creation of proteins, plasmids coding therefor and organisms containing such plasmids.

2. Background Details

It’s known to alter microbes to create desirable proteins, for example enzymes, by incorporating plasmids coding for a desirable protein to a microbe that would not otherwise create it which wouldn’t create it in sufficient quantity.There is a tendency for the plasmids to be dropped prolonged cultivation of this organism. It’s believed that this is at least partly as a result of production, on every branch of the microbes, of a few female cells that contain noneof the said plasmids and that can be in effect in a particular advantage over those that contain the desired plasmids. In the course of time, cells that contain none of the desirable plasmids growth as a proportion of their total cells present.

To be able to overcome this effect it is known to incorporate in the plasmid one or more genes that give a selective advantage to this microbe, for example genes giving resistance to an antibiotic are more suitable if the microbes are cultivated in thepresence of the antibiotic, or even enzymes which makes great a deficiency in the host organism might be incorporated.

There is also a trend for mutated variants of the plasmid which do not create the desired protein to be produced such as by a mutation leading to the introduction of a stop codon in the gene or even partial or complete deletion of the proteincoding sequence. Microbes containing such mutated plasmids will tend to have a selective advantage compared with people using the original plasmid and the desired protein generating capability of the microbe might be reduced or lost on prolongedcultivation.

In DDR patent 233,851 A1 there are disclosed vector plasmids where a sequence is present twice in opposite senses (inverted repeat sequences) into every one of which sequences duplicate genes are cloned or recloned. This is said to cause increasedsynthesis of this gene product in the microorganism on account of the gene dose effect. The inverted repeat sequences need to each have a minumum of one homologous cleavage site to that the duplicate genes could be inserted. Stable plasmids are disclosed asproducible.

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