Protease-deficient bacillus anthracis
Inventors
Pomerantsev, Andrei P. • Leppla, Stephen H.
Assignees
US Department of Health and Human Services
Publication Number
US-10273548-B2
Publication Date
2019-04-30
Expiration Date
2032-08-02
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Abstract
The invention relates to a Bacillus anthracis (B. anthracis) in which more than one secreted protease is inactivated by genetic modification. Such a protease-deficient B. anthracis has an improved ability to produce recombinant secreted proteins compared to other bacteria, particularly other Bacillus. Improvements include production of intact (i.e., mature full-length) proteins, often at high yield. The disclosure provides a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. Also provided is a modified B. anthracis comprising such genetic modification transformed with a recombinant molecule encoding a product, as well as methods to prepare and use such B. anthracis.
Core Innovation
The invention relates to a Bacillus anthracis (B. anthracis) strain genetically modified to inactivate multiple secreted proteases, improving its ability to produce recombinant secreted proteins, particularly intact mature full-length proteins, often at high yield.
Specifically, the invention provides B. anthracis strains comprising genetic modifications inactivating proteases from the M4 family of metalloproteases and the M6 family of metalloproteases, and optionally further inactivating proteases from the M73 family of metalloproteases and the ZnMc superfamily of zinc-dependent metalloproteases. Examples include inactivating NprB (M4), InhA1 and InhA2 (M6), camelysin and TasA (M73), and MmpZ (ZnMc). Additional proteases such as CysP1 (transglutaminase-like superfamily) and VpR (peptidase family S8) may also be inactivated. These protease-deficient strains are sometimes sporulation-deficient and may lack one or both virulence plasmids pXO1 and pXO2.
The problem addressed arises from the large quantities of extracellular proteases secreted by Bacillus species, including B. anthracis, which degrade recombinant proteins produced by the bacteria, reducing yield and integrity of proteins such as anthrax toxin components PA, EF, and LF. Existing strains with single protease inactivations still exhibit protein degradation, and Bacillus anthracis strains have not been effective generic hosts for recombinant protein production due to these protease activities.
The invention solves this problem by genetic modification to inactivate multiple proteases simultaneously, creating a protease-deficient B. anthracis that produces higher yields of intact recombinant proteins. Such strains, exemplified by BH460 and BH480, provide improved hosts for protein production, including production of highly active intact edema factor and other recombinant proteins.
Claims Coverage
The patent contains 15 claims focusing on genetically modified B. anthracis strains with various combinations of protease inactivations and phenotypic features.
Multi-protease knockout B. anthracis strain
A Bacillus anthracis strain comprising genetic modifications inactivating six specific proteases: NprB (locus GBAA_0599), InhA2 (GBAA_0672), TasA (GBAA_1288), Camelysin (calY, GBAA_1290), InhA1 (GBAA_1295), and MmpZ (GBAA_3159). The strain can be sporulation-deficient, lack virulence plasmids pXO1 and/or pXO2, or both.
Additional protease inactivation in B. anthracis
The above strain further genetically modified to inactivate proteases CysP1 (cysP1 gene, locus GBAA_1995) and VpR (vpR gene, locus GBAA_4584).
Further protease inactivation
The strain of previous features further genetically modified to inactivate protease NprC (nprC gene, locus GBAA_2183).
Expanded protease inactivation
The strain of prior features further genetically modified to inactivate protease SprA (sprA gene, locus GBAA_5414).
Additional protease inactivation with HtrA
The strain further genetically modified to inactivate protease HtrA (htrA gene, locus GBAA_3660).
Protease-deficient strain phenotypic feature
The strain is sporulation-deficient and lacks virulence plasmid pXO2.
Protease-deficient strain phenotypic feature with additional protease inactivations
Any of the strains further comprising CysP1 and VpR inactivations, being sporulation-deficient and lacking plasmid pXO2.
Protease-deficient strain phenotype and inactivations
Any of the strains further comprising NprC inactivation, being sporulation-deficient and lacking plasmid pXO2.
Protease-deficient strain with SprA inactivation and phenotype
Any of the strains further comprising SprA inactivation, being sporulation-deficient and lacking plasmid pXO2.
Protease-deficient strain with HtrA inactivation and phenotype
Any of the strains further comprising HtrA inactivation, being sporulation-deficient and lacking plasmid pXO2.
Protease-deficient strain phenotype lacking both virulence plasmids
The strain is sporulation-deficient and lacks virulence plasmids pXO1 and pXO2.
Strain with further protease inactivations and dual plasmid deletion
Any of the strains described above further comprising CysP1 and VpR inactivations, being sporulation-deficient and lacking plasmids pXO1 and pXO2.
Additional strain with NprC inactivation and dual plasmid deletion
As above, further comprising NprC inactivation, sporulation deficiency, and lacking pXO1 and pXO2.
Further protease and phenotype modification
Any of the strains further comprising SprA inactivation, sporulation deficiency, and lacking pXO1 and pXO2.
Further protease inactivation with HtrA and phenotype
Any of the strains further comprising HtrA inactivation, sporulation deficiency, and lacking pXO1 and pXO2.
The claims cover genetically modified B. anthracis strains with inactivated multiple extracellular proteases from specific families and genes, optionally deficient in sporulation and virulence plasmids, providing improved hosts for recombinant protein production.
Stated Advantages
Improved ability of B. anthracis to produce recombinant secreted proteins as intact mature full-length proteins often at high yield.
Increase in stability and yield of anthrax toxin proteins such as edema factor, protective antigen, and lethal factor.
Production of highly active recombinant edema factor from B. anthracis, overcoming previous degradation and low potency issues.
Sporulation-deficiency of strains reduces contamination risks.
Removal of virulence plasmids enhances safety and attenuates strains.
Documented Applications
Production of recombinant anthrax toxin proteins, e.g., protective antigen (PA), edema factor (EF), and lethal factor (LF).
Production of recombinant Bacillus cereus hemolysin HBL toxin components (L1, L2, B).
Production of heterologous recombinant proteins including toxins from other organisms, toxin fusion proteins, and tumor-targeted toxins.
Use of produced proteins for vaccines, therapeutics, diagnostics (e.g., antibody detection assays), and research reagents.
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