Wisconsin Alumni Research Foundation

Cellsnap LLC

Mcw Research Foundation, Inc.

Marite Bradshaw

Eric Arthur Johnson

William Howard Tepp

Sabine Pellett

Joseph T Barbieri

Kristin M Marshall

Michael Baldwin

Michael C Goodnough

Christina Pier

Dena Lyras

Julian I Rood

William H Tepp

Christina L Pier

Molly Moritz

Eric Johnson

The present disclosure relates to neurotoxins and uses thereof. In particular, provided herein are botulinum neurotoxins with altered properties and uses thereof (e.g., research, screening, and therapeutic uses).

Neurotoxins and uses thereof

The present disclosure relates to chimeric neurotoxins and uses thereof. In particular, provided herein are chimeric botulinum neurotoxins with altered properties and uses thereof (e.g., research, screening, and therapeutic uses).

Hybrid neurotoxins and uses thereof

A mutant strain of the bacteriumhaving an inactivated botulinal neurotoxin gene is disclosed. The mutant strain contains an artificially created and inserted modified intron vector between nucleotides 580 and 581 of the sense strand of the gene. The mutant strain can be used in microbiological challenge testing of foods and food processing methods.

[object Object]

The present invention provides novelconjugatively transmissible plasmids and methods of use thereof. Specifically, described herein are novel, conjugatively transmissible clostridial plasmids which are capable of being transferred among and between clostridial species. The novel plasmids of the present invention therefore permits the delivery of heterologous clostridial genes into a clostridial host, such as, and the expression of genes of interest in that host, including clostridial toxins and the nontoxigenic components of the toxin complex, toxin fragments, or antigenic portions thereof, in a way both that ensures abundant expression and facilitates purification. Furthermore, toxins with altered structures, chimeric, hybrid toxins, and other toxin derivatives valuable in medicine could be synthesized in this system.

Conjugative plasmids and methods of use thereof

The present invention provides a novel isolated plasmid, wherein the plasmid is a native plasmid found in uniquetype A strains and encode either BoNT/A3 or BoNT/A4 and BoNT/B. The present invention also provides a method of obtaining a plasmid-encoded botulinum neurotoxin and botulinum neurotoxin complex comprising the step of isolating a plasmid encoding the cntA/A or cntA/B neurotoxin gene and genes encoding protein components of the toxin complex from atype A strain. The inventors performed comparative analyses of representative BoNT/A subtype strains by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations with probes specific for the BoNT/A and B genes, cntA/A and cntA/B. Unexpectedly, the inventors determined that the genes encoding BoNT/A3 in the A3 strain, and BoNT/A4 and BoNT/B in the A4 strain, are on plasmids.

The present invention provides a novel isolated plasmid, wherein the plasmid is a native plasmid found in uniquetype A strains and encode either BoNT/A3 or BoNT/A4 and BoNT/B. The present invention also provides a method of obtaining a plasmid-encodedneurotoxin andneurotoxin complex comprising the step of isolating a plasmid encoding the cntA/A or cntA/B neurotoxin gene and genes encoding protein components of the toxin complex from atype A strain. The inventors performed comparative analyzes of representative BoNT/A subtype strains by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations with probes specific for the BoNT/A and B genes, cntA/A and cntA/B. Unexpectedly, the inventors determined that the genes encoding BoNT/A3 in the A3 strain, and BoNT/A4 and BoNT/B in the A4 strain, are on plasmids.

The present invention provides a preparation of botulinum toxin light chain type A or E, wherein the preparation is both catalytically active and soluble. Preferably, the preparation consists essentially of amino acid residues 1 through 425 of the botulinum toxin light chain type A. A method of screening inhibitors is also provided, wherein the method comprises exposing a test inhibitor to the preparation of botulinum toxin light chain type A and evaluating the biological activity of the preparation. In another embodiment, a method of providing a catalytically active, soluble preparation of botulinum toxin light chain, type A is provided, wherein the method comprises obtaining an expression vector comprising a DNA sequence encoding amino acid residues 1-425 and expressing a polypeptide.

Method of preparing botulinum neurotoxin type E light chain

A method of producing botulinum toxin C-terminal receptor binding domain (HCR) is disclosed. The one embodiment, the method comprises the steps of (a) preparingtransformed with an expression vector comprising DNA encoding HCR protein, (b) inducing expression of the HCR protein at a reduced temperature in a culture media, and (c) purifying the HCR protein via extraction, wherein the extraction comprises a clarification by centrifugation and a filtration, wherein the purified HCR protein is at least 10 mg/L of culture medium.

Method for preparing botulinum neurotoxin type A light chain

A hybrid botulinal neurotoxin is disclosed. In one embodiment, the neurotoxin comprises a combination of a botulinal neurotoxin heavy chain and light chain, wherein the light chain and heavy chain are not of the same serotype and wherein the heavy and light chains are linked by a homobifunctional sulfydryl linker. A method for creating hybrid neurotoxins comprised of different functional domains is also disclosed.

Hybrid botulinal neurotoxins

A system is used to express clostridial gene constructions in a clostridial host. A mobilizable transfer plasmid is described which permits the direct transfer of the plasmid, and genes carried on it, from E. coli into Clostridium species. A promoter is described for use in clostridial species. Also, a useful host strain is used which is nontoxigenic and which permits high levels of expression of clostridial genes using the clostridial promoter.

Expression system for clostridium species

A hybrid botulinal neurotoxin is disclosed. In one embodiment, the neurotoxin comprises a combination of a botulinal neurotoxin heavy chain and light chain, wherein the light chain and heavy chain are not of the same serotype. A method for creating hybrid neurotoxins comprised of different functional domains is also disclosed.

Growing community of inventors

Madison, WI, United States of America

Marite Bradshaw