Promoters for increased protein expression in meningococcus

Abstract

New promoters are described to drive transcription in meningococcus e.g. for over-expression of protein antigens for retention in membrane vesicles. Modified porA promoters lack the wild-type poly-G sequence which can cause phase variation. Meningococcal rRNA-coding genes (e.g. for 16S rRNA) can be used to drive transcription of a protein-coding gene. These approaches can be used in combination.

Claims

1. A nucleic acid comprising a promoter operably linked to a heterologous coding sequence, wherein the promoter is selected from the group consisting of: (a) a promoter comprising (i) a 10 region from a meningococcal porA gene promoter comprising TATAAT and (ii) a 35 region comprising the nucleic acid sequence TGGTTT or the nucleic acid sequence TTGACA, wherein the 10 region and the 35 region are separated by an intervening sequence comprising 12-20 nucleotides, wherein the intervening sequence either comprises no poly-G sequence or includes a poly-G sequence comprising no more than five consecutive G nucleotides; and (b) a promoter comprising a sequence selected from the group consisting of (i) the sequence of SEQ ID NO: 18 and (ii) a variant of SEQ ID NO: 18 which differs from SEQ ID NO: 18 by up to 4 single-nucleotide insertions, deletions, or substitutions.

2. The nucleic acid of claim 1, wherein the promoter of (a) has a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.

3. The nucleic acid of claim 1, wherein the intervening sequence does not comprise a sequence GGGGG.

4. The nucleic acid of claim 1, wherein non-transcribed sequence downstream of the 10 sequence and upstream of the transcription start site comprises 5-10 nucleotides.

5. The nucleic acid of claim 1, wherein the promoter of (a) comprises the sequence of SEQ ID NO: 30.

6. The nucleic acid of claim 1, wherein the promoter of (b) is selected from the group consisting of: nucleotides 6-39 of SEQ ID NO: 36, nucleotides 6-38 of SEQ ID NO: 37, and nucleotides 6-39 of SEQ ID NO: 38.

7. A bacterial expression vector comprising a DNA sequence comprising the nucleic acid of claim 1.

8. A meningococcus comprising a DNA sequence comprising the nucleic acid of claim 1.

9. The meningococcus of claim 8, wherein the heterologous coding sequence encodes an outer membrane protein.

10. The meningococcus of claim 9, wherein the outer membrane protein is a fHbp.

11. The meningococcus of claim 8, wherein the meningococcus does not express an active MltA.

12. The meningococcus of claim 8, wherein the meningococcus comprises a knockout of at least one of SynX and LpxL1.

13. The meningococcus of claim 8 comprising a meningococcus selected from the group consisting of serogroup B and serogroup W135.

14. The meningococcus of claim 8 comprising a lipopolysaccharide of immunotype L3.

15. The nucleic acid of claim 4, wherein the non-transcribed sequence comprises a sequence selected from the group consisting of TGAAGAC and TCGCAAC.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates fHbp expression levels in the same background strain (NZ98/254) using five different promoters and the benchmark strain overexpressing the same fHbp allele in the same background [112,113]. A variant 2 allele of fHbp was overexpressed in the benchmark strain or from either 2 wildtype phase variants of the porA promoter with either 11 Gs or 13 Gs in the spacer tract or with the modified ST1, ST2 and ST3 porA promoters as indicated. Western blotting of equivalent quantities of total protein were loaded from 0.5 to 2 as indicated for each strain, and the arrow shows fHbp.

(2) FIG. 2 illustrates comparative analysis of fHbp expression from porA and 16S modified promoters. The NZ98/254 strain expressing fHBP variant 2 under the control of the indicated promoters was grown on plates (A) and in liquid to logarithmic (B) and stationary phase (C) and levels of fHBP expression were measured by western blot from duplicate cultures (#1, #2) or from the benchmark overexpression strain. The unbroken arrow shows fHbp, whereas the lower broken arrow shows a loading control.

(3) FIG. 3 shows expression of fHbp from three strains: (a) a lpxL1 knockout; (b) a synX knockout; and (c) a lpxL1synX double knockout. In all cases the knocked out gene(s) was replaced by fHbp under the control of a modified PorA promoter. Expression of fHbp was assessed by western blot on crude cell extracts using anti-fHbp serum, and was compared to Hfq as a control. FIG. 3A shows the western blot, and FIG. 3B quantifies expression in arbitrary units.

(4) FIG. 4 shows fHbp levels in vesicles isolated from (i) lpxL1synX and (ii) lpxL1synXmltA knockout strains. FIG. 4A shows yield in mg/OD, and FIG. 4B is a western blot of 0.5 g vesicles.

(5) FIG. 5 shows expression of fHbp in two strains that overexpress either variant 1 or variant 2 fHbp in distinct loci (lanes c & d) or a strain that overexpresses both variant 1 and variant 2 in respective loci (lane e). Lane a is the wild-type strain, and lane b is a fHbp knockout of that strain.

(6) FIG. 6 shows silver-stained SDS-PAGE of vesicles purified from the triple knockout.

MODES FOR CARRYING OUT THE INVENTION

Modified PorA Promoters

(7) The sequences upstream of the transcription start site of two phase variants of the wild-type meningococcal porA promoter, containing 11 and 13 consecutive Gs in the spacer region, are as follows, with the 35 and 10 regions underlined:

(8) TABLE-US-00001 (SEQIDNO:31) AAAAATGGTTTTTTGCGGGGGGGGGGGTATAATTGAAGAC (SEQIDNO:40) AAAAATGGTTTTTTGCGGGGGGGGGGGGGTATAATTGAAGAC

(9) The intervening sequence between the 35 and 10 regions includes a G.sub.11 poly-G sequence which is connected to phase variation of PorA expression. To stabilise expression from this promoter, three modifications were tested (modifications in lower case):

(10) TABLE-US-00002 (ST1;SEQIDNO:32) AAAAATGGTTTTTTGCGaGGGaGGtGGTATAATTGAAGAC (ST2;SEQIDNO:33) AAAAATtGacaTTTGCGaGGGaGGtGGTATAATTGAAGAC (ST3;SEQIDNO:34) AAAAtTGacaTTTTGCGaGGGaGGtGGTATAATTGAAGAC

(11) Thus in ST1 the G.sub.11 sequence was interrupted in three places, leaving no more than 3 consecutive G residues. In ST2 the same intervening sequence was used, but the 35 region was substituted by a consensus 35 region TTGACA. In ST3 an extra T residue was inserted immediately downstream of the 35 region.

(12) These three promoters were used to drive expression of a fHbp sequence (fHbp variant 2) in the NZ98/258 strain. All three modified promoters resulted in high level overexpression of fHBP in the strain when compared to an equivalent overexpressing strain which has been reported in the literature [112,113]. The best expression was seen with ST2 (FIGS. 1 & 2).

(13) Modified rRNA Promoters

(14) In further experiments the 16S rRNA promoter was fused to the 5 UTR of the porA gene:

(15) TABLE-US-00003 (SEQIDNO:35) ATATCTTGACAGGCGGAAGGAATACTTTATAATTCGCAAC...

(16) This promoter was subjected to mutation and three variants downstream of the 35 region were sequenced as follows:

(17) TABLE-US-00004 (wt;SEQIDNO:35) ATATCTTGACAGGCGGAAGGAATACTTTATAATTCGCAAC (#5;SEQIDNO:36) ATATCTTGACAGGCGGAAGGAATACTTTATATTCGCAAC... (#6;SEQIDNO:37) ATATCTTGACAGGCGGAAGGAATACTTTTAATTCGCAC... (#8;SEQIDNO:38) ATATCTTGACAGGCGGAAGGAAACTTTATAATTCGCAAC...

(18) Thus in variants #5 and #6 the 10 sequence is a 5-mer, and variant #6 also lost a nucleotide between the 10 region and the transcription start site. In variant #8 the 10 region and its downstream sequence are the same as wt, but the intervening sequence between the 35 and 10 regions is one nucleotide shorter than wt.

(19) Expression of fHbp from Modified Promoters

(20) These promoters were fused to the 5UTR region of the porA gene and used to drive expression of the fHbp coding sequence. As seen in FIG. 2, variants #5 and #6, variants #5 and #6 showed the strongest expression, approaching levels seen with ST2.

(21) The complete region spanning the wild-type promoter (SEQ ID NO: 35), extending downstream into the transcribed region and also upstream, was as follows (SEQ ID NO: 41; 358mer), with the 35 and 10 regions underlined, and nucleotide +1 (following reference 10) double-underlined:

(22) TABLE-US-00005 (SEQIDNO:41) ...CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGGAAAG GATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATTTCCC CAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCTGCAG AAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCGGAAG GAATACTTTATAATTCGCAACGTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATCAAATTTACAAAAG GAAGCC...

(23) Similarly, the surrounding sequences for variants #5 and #6 were as follows:

(24) TABLE-US-00006 (SEQIDNO:42) #5:...CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGG AAAGGATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATT TCCCCAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCT GCAGAAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCG GAAGGAATACTTTATATTCGCAACGTATCGGGTGTTTGCCNNANGTTTTTAGGTTTTTATCAAATTTCAAA AGGAAGCC... (SEQIDNO:43) #6:...CGTCTGAGTCCCCGAGTTTCAGACAGCATATTCACAAAGGCGCACCAGCCGGAGGAGGGAGAGG AAAGGATTGTTGGAGGCGGCGCAGTATTTAGCAGAAATAAAAAACCTTATCCGACAGCGACATGACGAATT TCCCCAAAAAAATCCCGCTGAAAGCATTGACCGTTTTTCCCTGTGGGCGTATAGTTCGGTTCTTCGCTGCT GCAGAAGTGGCGGACGAACTGAAAAGTATAGCACAGAATGTTGGGGATATCGAGAGATATCTTGACAGGCG GAAGGAATACTTTTAATTCGCACGTATCGGGTGTTTGCCCGATGTTTTTAGGTTTTTATTAAATTTACAAA AGGAAGCCCATANGAATCGAACTGC...

(25) Any of these longer sequences can be used with the invention, although modifications as discussed herein may, of course, be made.

(26) A fHbp gene under the control of a variant promoter was stably inserted into the chromosome of a meningococcus (strain NZ98/254) in place of the endogenous lpxL1 and/or synX gene(s). Slightly higher expression levels were seen at the synX locus, and expression for the strain with both insertions (lpxL1synX double knockout) was the sum of the expression from the individual loci (FIG. 3).

(27) Strains were also made in which two different fHbp variants (1 & 2) were expressed under the control of a modified PorA promoter. The co-expression of both variants has no significant negative effect on the expression from each distinct locus, and instead resulted in an additive effect (FIG. 5).

(28) Knockout of the endogenous mltA (GNA33) gene in the lpxL1synX double knockout further increased expression levels, and had no negative impact on the localisation of fHbp protein to the strain's vesicles. FIG. 4 shows fHbp levels in vesicles isolated from these strains, showing (A) the yield of protein (mg) relative to the culture's optical density (OD), and (B) an anti-fHbp western blot against of 0.5 g of the vesicles. SDS-PAGE shows that the fHbp band in the triple knockout strain was one of the most abundant proteins in the vesicles, along with PorA and PorB (FIG. 6).

(29) It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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