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
2000-04-04
2002-08-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S183000, C435S252300, C435S320100
Reexamination Certificate
active
06428981
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a Bacillus cell for improved production of a polypeptide of interest and a process for producing a polypeptide of interest.
2. Description of the Related Art
Bacillus cells have been widely used for industrial production of polypeptides of interest. Kunst et al., Nature 390: 249-256 (1997) describes the complete genome sequence of the Gram-positive bacterium
Bacillus subtilis.
SUMMARY OF INVENTION
The problem to be solved by the present invention is to provide a Bacillus cell capable of producing increased yields of a polypeptide of interest.
The solution is based on that the present inventors have identified that a Bacillus cell expressing less than wild-type level of a gene comprising the DNA sequence shown in SEQ ID NO:1 produces increased yields of a polypeptide of interest.
The gene is identified in a
Bacillus subtilis
cell and is termed yjbH in the
Bacillus subtilis
cell. See Kunst et al., Nature 390:249-256 (1997).
Further, the inventors have identified a homologous yjbH gene in a
B. licheniformis
cell (SEQ ID NO:3).
As stated above (see Background; Kunst et al., Nature 390: 249-256 (1997)) the yjbH gene was, at the priority date of the present invention, NOT annotated, i.e. no known function had been associated with the gene. Based on the teaching provided herein it is among the skilled persons' general knowledge to identify a homologous gene in another Bacillus cell, e.g. by DNA sequence homology to the yjbH genes disclosed herein (SEQ ID NO:1 and SEQ ID NO:3) and thereby readily be able to produce a Bacillus cell capable of producing an increased yield of a polypeptide of interest according to the solution outlined above.
Further, the DNA sequence shown in SEQ ID NO:1 has very low identity to any other known DNA sequences from any cell. A homology search performed in the publicly known databases such as EMBL, showed that no other sequence had any close identity to the DNA sequence shown in SEQ ID NO:1.
Likewise a homology search in the publicly available SWISSPROT database, using the polypeptide sequence shown in SEQ ID NO:2, showed no other polypeptide with any close identity.
Accordingly, in a first aspect the present invention relates to a Bacillus cell for improved production of a polypeptide of interest, wherein the Bacillus cell expresses less than wild-type levels of a gene that comprises:
(a) the DNA sequence shown in positions 1 to 828 in SEQ ID NO:1;
(b) a DNA sequence which is at least 70% identical to the DNA sequence of item (a);
(c) a DNA sequence which encodes a polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2; or
(d) a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2.
Further, the present inventors have identified that a partial sequence shown as DNA sequence from position 1 to 147 in SEQ ID NO:1 is highly conserved.
Accordingly, in a second aspect the present invention relates to a Bacillus cell for improved production of a polypeptide of interest, wherein the Bacillus cell expresses smaller than wild-type amounts of a gene that comprises:
(a) the DNA sequence shown in positions 1 to 147 in SEQ ID NO:1;
(b) a DNA sequence which is at least 70% identical to the DNA sequence of item (a);
(c) a DNA sequence which encodes a polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2; or
(d) a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2.
As it is clear from the above a Bacillus cell as described herein is highly suitable for the production of a polypeptide of interest.
Accordingly, a third aspect of the present invention is a process for producing a polypeptide of interest comprising the following steps:
(i) cultivating a Bacillus of the first or second preceding aspects under conditions permitting production of the polypeptide of interest;
(ii) isolating the polypeptide of interest.
Definitions
Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.
The term “a gene” denotes herein a gene (a DNA sequence) which is capable of being expressed into a polypeptide within said cell. Accordingly, this gene will be defined as an open reading frame starting from a start codon (normally “ATG”, “GTG”, or “TTG”) and ending at a stop codon (normally “TAA”, “TAG” or “TGA”)).
In order to express the gene there must be elements, as known in the art, in connection with the gene, necessary for expression of the gene within the cell. Such standard elements may include a promoter, a ribosomal binding site, a termination sequence, and may be other elements as known in the art.
The term “the Bacillus cell expresses less than wild-type levels of a gene” according to the first and second aspects of the invention denotes any alterations of the wild-type cell giving rise to a cell which expresses less than wild-type levels of a gene. These alterations may be alterations of a promoter and/or an open reading frame such as deletions, insertions, frame shifts or any manipulations of the DNA as known in the art.
The expression level of a gene in a Bacillus cell altered according to the above is preferably determined by comparing production levels of the polypeptide of interest in said cell with the production level of the polypeptide of interest in the parent non-altered Bacillus cell. If the altered cell produces more of the polypeptide of interest when compared to the non-altered cell, then the Bacillus cell, according to the first and second aspects of the invention, expresses less than wild-type levels of a gene. The actual assay for the determination of production levels of the polypeptide of interest will depend on the specific polypeptide of interest. It is among the skilled persons' general knowledge to choose the appropriate assay.
Identity of DNA Sequences
The DNA sequence identity in relation to the terms “a DNA sequence which is at least 70% identical to the DNA sequence shown in positions 1-828 of SEQ ID NO:1” of the first aspect and “a DNA sequence which is at least 70% identical to the DNA sequence shown in positions 1-147 of SEQ ID NO:1” of the second aspect of the invention is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the analogous DNA sequences referred to above exhibits a degree of identity preferably of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97% with the DNA sequence shown in positions 1-828 of the first aspect of the invention; or the DNA sequence shown in positions 1-147 of SEQ ID NO:1 of the second aspect of the invention.
Identity of Polypeptide Sequences
The polypeptide sequence identity in relation to the terms “a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2” of the first aspect and
“a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2” of the second aspect of the invention is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of co
Christensen Christina Lund
Joergensen Steen Troels
Kristensen Tina
Lambiris Elias
Novozymes A/S
Rao Manjunath N.
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