Novel Bacterial Expression System
The present invention relates to expression cassettes suitable for the expression of a heterologous gene. Furthermore, the invention relates to recombinant plasmids comprising such expression cassettes. Also, the invention relates to host cells comprising such cassettes or plasmids. Another embodiment relates to promoters for use in an expression cassette and in heterologous expression systems. Finally, an embodiment relates to methods for the expression of a heterologous gene.
1 . An expression cassette, characterized in that said expression cassette comprises a promoter obtainable from the region between the stop codon of the yyaF gene and the start codon of the rpsf gene as found on the Bacillus subtilis genome.
2 . An expression cassette, characterized in that said expression cassette comprises a promoter having the following characteristics
a) a nucleotide sequence TATAAT, preferably GTATAAT or TATAATA at the −10 site,
b) a nucleotide sequence TTGTAA, preferably GTTGTAA or TTGTAAA at the −35 site,
c) a spacer of 17+/−2 nucleotides, preferably 17+/−1 nucleotides, more preferably 17 nucleotides.
3 . The expression cassette according to claim 1 , characterized in that the promoter comprises the nucleotide sequence as depicted in SEQ ID NO 1.
4 . The expression cassette according to claim 1 , characterized in that the promoter comprises the nucleotide sequence as depicted in SEQ ID NO 2.
5 . The expression cassette according to claim 1 , characterized in that the promoter comprises the nucleotide sequence as depicted in SEQ ID NO 3.
6 . The Expression cassette according to claim 1 , characterized in that the promoter is preceded by a stretch of nucleotides having at least 1, preferably 2, more preferably even three AT-rich regions upstream of the −35 region.
7 . The expression cassette according to claim 6 , characterized in that said stretch of nucleotides having AT-rich regions upstream of the −35 region comprises the nucleotide sequence as depicted in SEQ ID NO 4, preferably in SEQ ID NO 5, more preferably in SEQ ID NO 6.
8 . An expression cassette, characterized in that said expression cassette comprises a promoter having a consensus sequence AAAANNNNNTTTTX n AAAANNNNNTTTT (SEQ ID NO: 16), wherein 10<n<150.
9 . The expression cassette according to claim 8 , characterized in that the promoter comprises the nucleotide sequence as depicted in SEQ ID NO: 7, preferably in SEQ ID NO: 8, more preferably in SEQ ID NO: 9.
10 . Recombinant plasmid characterized in that said plasmid comprises an expression cassette according to claim 1 .
11 . Bacterial host cell comprising an expression cassette according to claim 1 or a recombinant plasmid according to claim 10 .
12 - 14 . (canceled)
15 . A method for the heterologous expression of a gene, characterized in that said method comprises the transfection of a bacterium with an expression cassette according to claim 1.