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
1991-12-19
2001-09-04
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...
C435S252310, C435S252500
Reexamination Certificate
active
06284490
ABSTRACT:
The present invention relates to the asporogenous strain
Bacillus subtilis
SMS275 CMS 432.90 and its use as a host in a host-vector system for the preparation of heterologous products.
It is known in the art to prepare proteins and polypeptides by fermentation methods which use certain production systems, where this term means the combination of an expression vector containing the gene which codes for the protein or the polypeptide and the host which contains it.
Most recombinant products currently on the market are produced with the use of production systems which use
Escherichia coli
, CHO cells, or
Saccaromyces cerevisiae
as hosts.
However, these systems are not completely satisfactory and there is therefore a need in the art to provide other production systems for the preparation of heterologous products based, fundamentally, on the use of host organisms such as, for example, bacteria like Bacillus and Streptomyces or yeasts and fungi like
Kluyveromyces lactis
and
Yarrowia lipolytica
or insect cells.
From an industrial point of view, an ideal production system should enable the preparation of a recombinant product which can be purified easily and which has biological activity identical to that of the natural protein with high yields and at economically attractive costs.
Such a system should be constituted by:
1) a vector which contains strong regulation elements (promoter and terminator), of which many copies are present in the cells, and which is conserved within the cells in a stable manner so as to produce a high yield of the product of interest, and
2) a host which correctly carries out the instructions provided for the heterologous gene so as to enable the production of a product which is identical to the natural product, and which is suitable for culture on a commercial scale, that is, which is resistant, can multiply to high densities, is not very demanding as regards its need for nutritive elements, and is safe, that is, does not produce toxic contaminants.
At the moment there is considerable interest in the development of expression systems for the production of recombinant products in
Bacillus subtilis
(
B. subtilis
).
In fact
B.subtilis
is a particularly attractive micro-organism from a biotechnological point of view because of its completely non-pathogenic nature, its ability to secrete the gene-expression product in the culture medium and, finally, because of the ease with which it can be grown on a large scale.
The use of
B.subtilis
as a host for the expression of heterologous proteins and polypeptides of interest to the pharmaceutical and food industries may thus be a determining factor for the approval of processes for the production of these products.
A partial contraindication with regard to the use of the micro-organism results, however, from its ability to give rise to the formation of spores under certain physiological growth conditions.
In fact spores, which are characterised by high resistance to chemical-physical agents have a high probability of survival under most normal environmental conditions.
The use of a recombinant strain of
B.subtilis
in a method of producing products of interest in the pharmaceutical and food fields may consequently encounter legal obstacles if it is not shown that there is a low probability of any spores generated being dispersed into the outside environment.
The formation of endospores in
B.subtilis
is a cell-differentiation process which takes place by means of sequential changes in the physiology of the cells and in their ultrastructure as a result of a response to conditions in which growth nutrients are restricted.
During sporulation which, on average, takes from 6 to 8 hours at 37° C., the cell passes through a series of well-defined morphological stages which terminate with the formation, within the sporangium, of an alternative cell form to the vegetative form, the endospore.
These stages, which are conventionally defined as stages (0-VII), require a series of substances which are coded by different genes, the spo genes.
The preparation of strains of
B.subtilis
which do not produce spores (asporogenous strains) by the mutation of a spo gene by chemical or physical agents or with the use of mutagenesis techniques in vitro is known in the art.
The mutants, which can no longer bring the formation of the spores to maturity, can in theory be used to prepare heterologous proteins.
Some of the mutants, however, have been found not to be completely satisfactory hosts for the development of systems for the asporogenous expression of
B.subtilis
because of the instability of the spo
−
phenotype, because of their inability to conserve the expression vector in a stable manner, and finally, because of the small number of copies of the vector present in the strain.
An asporogenous mutuant of
B. Subtilis
which overcomes the problems described above has now been isolated. This mutant, known as SMS275, has been deposited on Oct. 5, 1990 at the Centraalbureau Voor Schimmelcultures, Oosterstraal Postbus 273, NL-3740 AG Baarn Netherlands where it received the number CBS 432.90.
A subject of the present invention is therefore the asporogenous strain
Bacillus subtilis
SMS 275.
A further subject of the present invention is the use of the strain as a host in a host-vector system for the production of heterologus products.
Another subject of the present invention is a method for the preparation of a heterologous product of interest, including the transformation of the asporogenous strain
Bacillus subtilis
SMS 275 by an expression vector containing the gene which codes for the heterologous product, the growth of the transformed strain
Bacillus subtilis
SMS 275 in a suitable culture medium and, finally, the separation and purifcation of the gene-expression product thus produced.
In particular, the asporogenous strain
Bacillus subtilis
SMS 275 according to the present invention is characterised by the genetic markers spoII:D
−
, leu (an inability to grow in minimal medium in the absence of leucine), pyrD1 (an inability to grow in minimal medium in the absence of uracil), apr
−
and npr
−
(the inability to produce serinic protease and neutral protease).
The strain can also conserve the spo
−
phenotype in a stable manner, actually reverting to the formation of spores with a frequency of less than about 10
−8
, and can conserve a large number of copies of a replicable expression vector in a stable manner.
The strategy used to construct the asporogenous strain according to the present invention consists of the mutation of a sporogenous strain of
B.subtilis
by a transposon and the isolation of the asporogenous mutants thus produced.
Transposons are elements of DNA which can be moved and inserted at different points in the genome, conferring new hereditary properties to the host strain. In fact, after insertion at points in the genome, as well as interrupting the sequence of a gene, which is manifested by a phenotypic mutation, transposons, which contain genes which code for resistance to antibiotics, confer to the host resistance to a particular antibiotic.
According to one embodiment of the present invention, the transposon TN917 (Tomich and Clewell, (1980), J. Bacteriol., 141: 1366-1574), which, amongst other things, codes for resistance to the antibiotic erythromycin (Em), is used.
Mutation by the insertion of a transposon can be carried out by conjugation or transformation according to known techniques.
In particular, the asporogenous strain according to the present invention was produced by transforming the wild type (sporogenous) strain
B.subtilis
SMS 118 with a plasmid which is not replicable in Bacillus and which contains the transposon TN917 and selecting the mutated strains on a medium supplemented with erythromycin.
In fact, in theory, only the clones in which the transposon TN917 has been integrated into the chromosomal DNA of the sporogenous strain can grow on this medium.
Plasmids suitable for the purpose may be, for example, pTV1TS, pTV32TS, or pTV51TS (You
Cosmina Paola
Frascotti Gianni
Grandi Guido
Eniricerche S.p.A.
Gansheroff Lisa
Ketter James
Sughrue Mion Zinn Macpeak & Seas, PLLC
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