Production of proteins, plasmids coding therefor and organisms c

Details Production of proteins, plasmids coding therefor and organisms c Production of proteins, plasmids coding therefor and organisms c

1998-08-21

2000-10-24

Yucel, Remy

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

485 691, 485 711, 485183, 4852523, 48525233, 4853201, 485440, 485471, 485476, C12P 2104

Patent

active

06136567&

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
THIS INVENTION relates to the production of proteins, plasmids coding therefor and organisms containing such plasmids.
2. Background Information
It is known to modify microbes to produce desired proteins, for example enzymes, by incorporating plasmids coding for a desired protein into a microbe which would not otherwise produce it or which would not produce it in sufficient quantity. There is however a tendency for the plasmids to be lost on prolonged cultivation of the organism. It is believed that this is at least partly due to the production, on each division of the microbes, of a small number of daughter cells which contain none of the said plasmids and which are in consequence at a selective advantage over those which contain the desired plasmids. In the course of time, cells which contain none of the desired plasmids increase as a proportion of the total cells present.
In order to overcome this effect it is known to incorporate in the plasmid one or more genes which give a selective advantage to the microbe, for example genes giving resistance to an antibiotic are suitable if the microbes are cultivated in the presence of the antibiotic, or genes making good a deficiency in the host organism may be incorporated.
There is also a tendency for mutated variants of the plasmid which do not produce the desired protein to be produced for example by a mutation leading to the introduction of a stop codon in the gene or partial or complete deletion of the protein coding sequence. Microbes containing such mutated plasmids will tend to have a selective advantage compared with those having the original plasmid and the desired protein producing capability of the microbe may be reduced or lost on prolonged cultivation.
In DDR patent 233,851 A1 there are disclosed vector plasmids in which a sequence is present twice in opposite senses (inverted repeat sequences) into each of which sequences duplicate genes are cloned or recloned. This is said to cause increased synthesis of the gene product in the microorganism due to the gene dosage effect. The inverted repeat sequences must each have at least one homologous cleavage site into which the duplicate genes may be inserted. Stable plasmids are disclosed as producible.


SUMMARY OF THE INVENTION

Surprisingly we have now found that according to this invention stable plasmids can be produced from vectors which do not have inverted repeat sequences. This has important advantages.
Firstly, it reduces or eliminates the number of homologous cleavage sites and therefore the tendency to cut the plasmid into ineffective fragments in the process of cutting it to insert the desired gene. It is clear that each inverted sequence of DDR 233851 A1 must have at least one cleavage site and that these will be homologous. Normally the vector plasmids will be cleaved at both such sites. This increases the difficulty of constructing a plasmid with the desired gene correctly inserted into both of the inverted sequences. For example the free ends may link with a single added gene leaving plasmids with only one such gene. In addition non-functional nucleic acid fragments will be formed. The greater the number of cleavage sites in the inverted sequences the greater this problem becomes.
Secondly, it reduces the dangers of inter- and intra-plasmid recombination which can lead to scission of the plasmid. The presence of unnecessary DNA adjacent to the desired genes increases the likelihood of recombination leading to the loss of part or all of that gene. In addition, recombination between the inverted repeat sequences in different plasmid molecules (copies) may lead to formation of unstable multimeric plasmids.
By "Expression cassette" is meant a DNA sequence effective in production of a protein which comprises a promoter sequence and ribosomal binding site, a gene coding for a protein and normally a termination sequence. The gene may code for a fusion protein and may have a signal sequence.
Thirdly, it enables the production of smaller plasm

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New England Biolabs Catalog 1995. New England Biolabs, Inc. Beverly, MA., pp. 216-217.
Kellett, et al: "Xylanase B and an arabinofuranosidase from Pseudomonas fluorescene subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes", Biochem. J. (1990) 272:369-376.
Barth, et al: "Cloning and Partial Sequencing of an Operon Encoding Two Pseudomonas putida Haloalkanoate Dehalogenases of Opposite Stereospecificity", J. Bacteriology (1992) 174:2612-2619.
Weyer, et al: "A baculovirus dual expression vector derived from the Autographa califonica nuclear polyhedrosis virus polyhedin and p10 promoters: co-expression of two influenza virus genes insect cells", Journal of General Virology (1991), 72: 2967-2974.
Caunt, et al: "Stability of recombinant plasmids in yeast", Journal of Biotechnology (1988) 8: 173-192.

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