Protease-deficient Bacillus anthracis
Inventors
Pomerantsev, Andrei P. • Leppla, Stephen H.
Assignees
US Department of Health and Human Services
Publication Number
US-9555064-B2
Publication Date
2017-01-31
Expiration Date
2032-08-02
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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) 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 species. The improvements include production of intact (mature full-length) proteins, often at high yield.
The disclosure provides B. anthracis strains comprising genetic modifications that inactivate one or more proteases from specific metalloprotease families, notably those of the M4 and M6 families. Further embodiments include inactivation of proteases of the M73 family and the ZnMc superfamily of zinc-dependent metalloproteases. Specific proteases such as NprB (M4 family), InhA1 and InhA2 (M6 family), camelysin and TasA (M73 family), and MmpZ (ZnMc superfamily) are inactivated by genetic modification, and combinations of these inactivations produce strains with progressively improved recombinant protein production. Additional genetic modifications include inactivation of other proteases such as CysP1 and VpR, removal of regulatory proteins SinR and SinI, sporulation deficiency, and removal of virulence plasmids.
The background problem addressed is that B. anthracis and related Bacillus species produce large quantities of extracellular secreted proteases that degrade recombinant and endogenous proteins produced, limiting yield and structural integrity of desired proteins such as anthrax toxin components. Existing strains producing recombinant proteins, such as anthrax edema factor and protective antigen, suffer from protein degradation due to these proteases. There remains an unmet need for B. anthracis strains engineered to lack multiple secreted proteases, enabling stable production of intact recombinant proteins in high yield.
Claims Coverage
The patent contains one independent claim covering a genetically modified Bacillus anthracis organism transformed with a recombinant molecule encoding a product. The claim covers inventive features related to specific protease inactivations and configurations of the organism to enhance production of intact recombinant proteins.
Protease-deficient Bacillus anthracis host
A Bacillus anthracis strain comprising genetic modifications that inactivate the proteases NprB, InhA2, TasA, camelysin, InhA1, and MmpZ, each encoded by genetically modified genes at specific loci on the B. anthracis genome.
Host strain genetic configurations
The B. anthracis is further characterized by one or more of the following: being sporulation-deficient, lacking virulence plasmids pXO1, pXO2, or both, or combinations thereof.
Recombinant molecule encoding an intact protein product
The B. anthracis is transformed with a recombinant molecule comprising a nucleic acid molecule encoding an intact protein product operatively linked to an expression vector suitable for expression in B. anthracis.
The claims cover a genetically modified Bacillus anthracis host with multiple inactivated protease genes and specific genetic backgrounds (sporulation deficiency and virulence plasmid deletions), transformed with recombinant molecules encoding intact protein products, especially toxins and anthrax toxin components. These features collectively enable improved production of intact recombinant proteins.
Stated Advantages
Protease-deficient B. anthracis strains produce intact (mature, full-length) recombinant proteins, often at high yield, compared to other Bacillus strains or strains with fewer protease deletions.
Deletion of multiple secreted proteases stabilizes anthrax toxin components and other recombinant proteins in culture supernatants over extended time, enhancing protein yield and integrity.
Specific strains such as BH460 and BH480 are sporulation-deficient, lack virulence plasmids, and produce highly active proteins with improved potency, such as edema factor with greater in vivo activity.
The modified strains offer improved hosts for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture, and in some cases yields exceeding 50 to 90 mg per liter.
Documented Applications
Production of stable, intact recombinant proteins including anthrax toxin components (protective antigen, edema factor, lethal factor), anthrolysin, Bacillus cereus hemolysins, and heterologous proteins.
Using protease-deficient, sporulation-deficient, and avirulent Bacillus anthracis strains for production of recombinant toxins or toxin fusion proteins, including for vaccine development and therapeutic use.
Methods of culturing genetically modified Bacillus anthracis strains transformed with recombinant molecules encoding desired protein products and recovering those proteins.
Use of Bacillus anthracis strains lacking specific proteases to produce proteins such as hemolysin HBL components, toxin fusion proteins like LFnBlaY, and variants of urokinase plasminogen activator.
Interested in licensing this patent?