Promoters

Details Promoters Promoters

2001-09-07

2004-08-03

Rao, Manjunath N. (Department: 1652)

Chemistry: molecular biology and microbiology

Vector, per se

C435S006120, C435S069100, C435S252300, C435S325000, C435S252500, C536S023100, C536S024100

Reexamination Certificate

active

06770475

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to promoters. More specifically, the present invention relates to novel promoters having restriction enzyme cleavage site sequences in the vicinity of the 3′ end downstream of the promoters and improved promoter activity.
2. Description of the Prior Art
To produce substances with microorganisms, the following operations are widely performed: inserting a gene encoding a desired protein into a vector and introducing the obtained recombinant vector into a cell to express this gene, thereby producing the desired protein in the host microorganism. One of the regions involved in the expression of the gene is a promoter.
The promoter includes a region including −35 and −10 regions which RNA polymerase recognizes and binds to and a region that designates the binding of ribosome to mRNA synthesized by RNA polymerase. Not only the nucleotide sequence of the promoter region is very important regarding expression efficiency, but the length of the nucleotide sequence thereof, that is, the number of bases, is also important (Mol. Gen. Genet. 186 339-(1982)).
Research of the nucleotide sequence of the promoter was first conducted with promoters of
E. coli
, leading to production of promoters for mass expression of proteins, and substance production using
E. coli
as a host has been performed.
However, since
E. coli
produces pyrogens, purification process in protein production is very expensive. Therefore,
E. coli
is not a suitable host for the fields of pharmaceuticals, foods, or the like, so that other host cells (microorganisms) are in demand. Among these, microorganisms of the genus Bacillus seem promising, because they have been used in fermentation industry, have high secretion performance, and are free from pathogenicity and do not produce pyrogens.
There are a number of reports regarding the production of protein using microorganisms of the genus Bacillus as a host. However, in most cases, the main purpose lies in ligating a gene encoding a desired protein to a secretory signal sequence in-frame for the purpose of secretion (e.g., Japanese Laid-Open Patent Publication (Tokkai) Nos. 59-205996, 62-215393, and 3-206889, Japanese Patent Publication (Tokko) No. 6-69377, and Japanese Laid-Open Patent Publication (Tokkai) No. 7-155183). Thus, there is substantially no attempt to enhance promoter activity.
Regarding improvement of the promoters of the genus Bacillus, Japanese Laid-Open Patent Publication (Tokuhyo) No. 6-500689 describes a hybrid promoter of &agr;-amylase of
Bacillus subtilis
, and Japanese Laid-Open Patent Publication (Tokuhyo) No. 7-504085 describes that a promoter having nine mutations at the 553
th
and from the 588
th
to 595
th
positions of an &agr;-amylase promoter gene of
Bacillus licheniformis
has higher activity than the natural sequence. However, the activity of the promoter does not appear to be sufficient.
Thus, there are substantially no reports on research results regarding improvement of promoters of the genus Bacillus. This seems be not because research has not been conducted, but because good results substantially cannot be attained. This seems to be one factor that prevents microorganisms of the genus Bacillus from being utilized as a recombinant host.
Therefore, in particular, in the field of pharmaceuticals or foods, it is desired at present to increase the expression efficiency of promoters of microorganisms of the genus Bacillus, and to increase the utility value of the microorganisms of the genus Bacillus as a host.
Therefore, it is an object of the present invention to provide highly active promoters of microorganisms of the genus Bacillus and to increase utilization of the microorganism of genus Bacillus as a host.
SUMMARY OF THE INVENTION
In order to solve the above problems, the inventors of the present invention found that promoter activity is improved by providing a mutation in a DNA sequence located in the vicinity of the 3′ end of an &agr;-amylase promoter region derived from
Bacillus amyloliquefaciens
and thus attained the present invention.
More specifically, the present invention is directed to a promoter of &agr;-amylase derived from a microorganism of the genus Bacillus, wherein a sequence having at least one restriction enzyme cleavage site (hereinafter, referred to as “restriction site”) is introduced between the vicinity of the 3′ end of the promoter and an initiation codon of protein, and the activity of the promoter is higher than that of a natural promoter.
In a preferable embodiment, the promoter of &agr;-amylase is derived from
Bacillus amyloliquefaciens.
In a preferable embodiment, the restriction site is a restriction site for BamHI.
In a more preferable embodiment, the promoter has the sequence of Sequence ID No. 1.
In another preferable embodiment, the restriction site includes restriction sites for BamHI and at least one restriction site other than the restriction site for BamHI, and the restriction site other than the restriction site for BamHI is present downstream of the BamHI restriction site.
In a more preferable embodiment, the restriction site sequence has a sequence of restriction sites for BamHI and EcoRI, and may have a sequence of at least one restriction site between the BamHI and EcoRI cleavage sites.
In an even more preferable embodiment, the sequence of the promoter is the sequence of Sequence ID No. 2, and the restriction sites are restriction sites for BamHI, SmaI, KpnI, SacI and EcoRI in this order from the 5′ end.
In another preferable embodiment, the restriction site sequence has a sequence of restriction sites for BamHI, NdeI, and XhoI in this order from the 5′ end.
The present invention is also directed to an expression cassette having the above-described promoter.
Furthermore, the present invention is also directed to an expression vector in which a gene encoding protein is inserted into a restriction site of this expression cassette.
In a preferable embodiment, the sequence encoding protein is a sequence of an intracellular enzyme.
In a more preferable embodiment, the sequence encoding protein is a sequence of phosphorylase or isomerase.
In an even more preferable embodiment, the phosphorylase is trehalose phosphorylase or maltose phosphorylase.
In another preferable embodiment, the isomerase is mannose isomerase.
The present invention is further directed to a recombinant microorganism having the above-described expression vector and a method for producing protein including the step of culturing this recombinant microorganism.


REFERENCES:
patent: 10-262683 (1998-10-01), None
patent: 10-327887 (1998-12-01), None
Palva et al. (Gene, 1981, vol. 15:43-51).*
Messing, J.,Methods in Enzymology, 101: 20-78 (1983), Academic Press, Inc.
Mandel, M., et al., “Calcium-dependent Bacteriophage DNA Infection”,J. Mol. Biol.53: 159-162 (1970).
Hanahan, D., “Studies on Transformation of Escherichia coli with Plasmids”,J. Mol. Biol.166: 557-580 (1983).
Contente, S. et al., “Characterization of Plasmid Transformation in Bacillus subtills: Kinetic Properties and the Effect of DNA Conformation”,Molec. gen. Genet.167: 251-258 (1979).
Chang, S. et al., “High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA”,Molec. gen. Genet.168: 111-115 (1979).

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