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
1999-02-26
2001-07-03
Prouty, Rebecca E. (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...
C435S320100, C435S325000, C435S252310, C435S252300, C435S183000, C435S201000, C435S209000, C435S207000, C435S198000, C435S194000, C435S195000
Reexamination Certificate
active
06255076
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for producing a polypeptide in a bacterial cell.
2. Description of the Related Art
Bacilli are well established as host cell systems for the production of native and recombinant proteins. However, Bacillus host cells with the desirable traits of increased protein expression may not necessarily have the most desirable characteristics for commercial use.
Conventionally, maximal expression of a gene contained in a Bacillus cell is achieved by amplifying in the chromosome an expression cassette containing a single promoter operably linked to a gene of interest and an amplifiable selectable marker gene, e.g., an antibiotic resistance marker. The amplification leads to the production of multiple copies of the expression cassette and the selectable marker gene in the chromosome.
However, there are disadvantages associated with this approach. For example, it may not be possible to achieve saturating levels of mRNA by amplifying genes driven by single promoters. Also, the production of multiple copies of the expression cassette and the selectable marker gene in the chromosome of a cell may not be stable. Furthermore, the removal of the selectable marker genes without also losing the expression cassettes may not be technically feasible.
Ichikawa et al. (1993
, FEMS Microbiological Letters
111: 219-224) disclose the extracellular production of cholera toxin B in
Bacillus brevis
containing an expression-secretiion vector with multiple promoters which mediate the expression of the gene encoding the mature cholera toxin B.
Agaisse and Lereclus (1994
, Molecular Microbiology
13: 97-107) disclose a structural and functional analysis of the promoter region involved in the full expression of the cryIIIA toxin gene of
Bacillus thuringiensis
. WO 94/25612 discloses an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the cryllIA gene which increases expression of the gene.
Hue et al. (1995
, Journal of Bacteriology
177: 3465-3471) disclose a 5′ mRNA stabilizer sequence which stabilized several heterologous RNA sequences when present at the 5′ end and increased expression of downstream coding sequences several-fold in
Bacillus subtilis.
It is an object of the present invention to provide improved methods for producing a polypeptide in a Bacillus strain.
SUMMARY OF THE INVENTION
The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a nucleic acid sequence encoding the polypeptide and alternatively also (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium.
The present invention also relates to Bacillus cells comprising a nucleic acid construct which comprises (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding a polypeptide and alternatively also (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide.
The present invention also relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium.
The present invention also relates to Bacillus cells comprising a nucleic acid construct which comprises (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding a polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide.
The present invention also relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid sequence encoding the polypeptide operably linked to a “consensus” amyQ promoter; and (b) isolating the polypeptide from the cultivation medium.
The present invention also relates to Bacillus cells comprising a nucleic acid construct which comprises (i) a “consensus” amyQ promoter operably linked to a single copy of a nucleic acid sequence encoding a polypeptide and (ii) an MnRNA processing/stabilizing sequence located downstream of the “consensus” amyQ promoter and upstream of the nucleic acid sequence encoding the polypeptide.
The present invention also relates to methods for producing selectable marker-free mutants of a Bacillus cell.
REFERENCES:
patent: WO 94/25612 (1994-11-01), None
Agaisse et al., Molecular Biology, 13 (1), pp. 97-107, (1994).
Hue et al., Journal of Bacteriology, vol. 177, No. 12, pp. 3465-3471, (1995).
Ichikawa et al., FEMS Microbiology Letters, 111, pp. 219-224, (1993).
Sloma Alan
Thomas Michael D.
Widner William
Monshipouri Maryam
Novozymes Biotech Inc.
Prouty Rebecca E.
Starnes Robert L.
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