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Proteins with Improved Solubility and Methods for Producing and Using Same

Inactive Publication Date: 2009-10-29
THE UK SEC FOR HEALTH & HER BRITANNIC MAJESTYS GOVERNMENT OF THE UK OF GREAT BRITAIN & NORTHERN IRELAND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]An object of the invention is to provide a method for reducing or preventing aggregate formation during purification and / or formulation of proteins, such as toxins and toxin fragments.
[0011]Another object of the invention is to provide greater batch-to-batch consistency within protein products when characterized by standard methods of protein analysis.
[0012]Still another object of the invention is to provide proteins with improved solubility and process characteristics.
[0013]Yet another object of the invention is to provide toxin proteins and toxin fragments with improved solubility and process characteristics for use as immunodiagnostic reagents, therapeutic agents, and vaccine components.
[0021]In one embodiment, then invention provides a recombinant protein comprising at least one point mutation that substitutes a cysteine residue with another amino acid residue, wherein said substitution improves the solubility of the recombinant protein.
[0059]In other embodiments, the invention provides a recombinant protein comprising a truncated botulinum serotype E toxin wherein the truncation improves the solubility of the recombinant protein.

Problems solved by technology

Aggregates tend to exhibit low solubility and other characteristics that are undesirable, such as low immunogenicity.
Its ingestion or inhalation inhibits neurotransmitter release from synaptic vesicles, resulting in neuroparalysis and death.
Although aggregated rLHN / E can be recovered from insoluble lysate material by detergent extraction / reductant treatment and further purified (˜90%) by anion exchange (Q Sepharose) and gel filtration (Superdex 200) chromatography, the recovered aggregate has undesirable properties.
Further, animal efficacy data indicate that immunization with aggregated LHN / E does not protect animals against BoNT / E toxin challenge.
Different fermentation conditions, for example, slow initial growth, less potent inducers, and / or reduced induction temperatures, as well as different detergent extraction / reductant treatments and denaturation (e.g. urea) / refolding methodologies have been being explored in an effort to produce soluble, non-aggregated or less aggregated LHN / E, but with limited success.

Method used

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  • Proteins with Improved Solubility and Methods for Producing and Using Same
  • Proteins with Improved Solubility and Methods for Producing and Using Same
  • Proteins with Improved Solubility and Methods for Producing and Using Same

Examples

Experimental program
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Effect test

example 1

Site-Directed Mutagenesis of LHN / E to Remove One or More Cysteine Residues

[0162]Endopeptidase-ablating mutations (E213Q and H216Y relative to SEQ ID NO: 1 and SEQ ID NO: 2) were introduced into the LHN / E coding sequence and the resulting cassettes cloned into plasmid vector pET26b. Various E. coli host strains were transformed and assessed for the ability to direct expression of a rLHN / E fragment. While high levels of target protein could be produced, recombinant LHN / E was expressed in all hosts as high molecular mass aggregate (FIGS. 1A and 1B). Those SDS-PAGE and gel filtration studies conducted under reducing and non-reducing conditions showed that LHN / E aggregation results, at least in part, from intermolecular disulfide bond formation.

[0163]It was hypothesized that aggregation could be due to the formation of cysteine-linked disulfide bonds between multiple LHN / E polypeptides. Molecular biology approaches were pursued to increase expression of soluble, non-aggregated rLHN / E pro...

example 2

Extending the LHN / E Fragment with Hc Sequence Improves Solubility

[0167]The solubility of clostridial neurotoxin proteins can also be enhanced by creating proteins in which an LHN domain, or a fragment of an LHN domain, is expressed along with amino acid sequence from an Hc domain.

[0168]Recombinant truncated forms of the botulinum serotype E toxin, such as the LHN fragments, have proven difficult to produce (express) and purify due to low solubility. Even at low concentrations, insoluble forms are often expressed in a non-native multimeric and aggregated state which renders them poor immunogens and unable to elicit protective levels of toxin neutralizing antibodies. To address this issue and enable the production of soluble (and possibly monomeric) protein, recombinant derivatives of the LHN / E protein have been produced that carry various lengths of the adjoining Hc domain.

[0169]The following expression constructs were tested for protein solubility in the E. coli strain ER2566: LHN / E...

example 3

Immunogenicity of LHN / E Cys to Ser Fragments and LHN / E-Hc Fragments

[0175]Abolishing the ability of LHN / E to form aberrant intermolecular disulfide bonds by replacing cysteine residues with amino acids that do not form disulfide bonds and extending the LHN / E fragment with Hc sequence are two techniques that improve the yield of monomeric or less aggregated protein. These modifications will enhance the immunogenicity and protective efficacy of the LHN / E fragment and the enzymatic activity of non-endopeptidase ablated toxins and toxin subfragments.

[0176]Immunogenicity of the recombinant proteins is tested in mice. Mice are immunized either with 10 μg of a LHN / E protein in which one or more cysteine residues has been replaced with another amino acid that does not form disulfide bonds, with 10 μg of a LHN / E protein that has been extended by inclusion of Hc sequence, with 10 μg of inactivated BoNT / E, or with other proteins described in the Examples. The proteins are suspended in an adjuva...

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Abstract

A method is provided for improving the solubility of proteins, for example, bacterial toxins. In one embodiment, solubility is improved by introducing point mutations that replace cysteine residues capable of forming intermolecular disulfide bonds with other amino acid residues that do not form such bonds. By abrogating the ability of the cysteine residues to form inter-molecular disulfide bonds, aggregation of the protein is reduced, thereby improving the solubility of the protein. In another embodiment, solubility of the protein is improved by producing truncated forms of the protein that express the LHN domain and a fragment of the Hc domain. Proteins made according to the method of the invention are useful, for example, as immunodiagnostic agents and vaccine components.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of U.S. provisional application Nos. 60 / 724,274, filed Oct. 7, 2005, and 60 / 742,900, filed Dec. 7, 2005, the entire disclosures of which are incorporated by reference.DESCRIPTION OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to methods for producing recombinant proteins that exhibit improved utility and process characteristics, such as solubility, compared to the corresponding native proteins. The invention also relates to proteins made according to the present methods, nucleic acids encoding the proteins, and the use of the proteins for prophylactic and therapeutic applications.[0004]2, Background of the Invention[0005]Proteins produced by organisms, and in particular microorganisms such as bacteria, are of interest because of their potential to serve as immunodiagnostic reagents, therapeutic agents, and vaccine components. Toxins are one group of proteins th...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K14/33A61P31/04C07H21/00C12N15/74A61K38/16C07K16/12
CPCC07K14/33A61K39/08A61P31/04Y02A50/30
Inventor SHONE, CLIFFORD C.CRAWFORD, JAMES A.
Owner THE UK SEC FOR HEALTH & HER BRITANNIC MAJESTYS GOVERNMENT OF THE UK OF GREAT BRITAIN & NORTHERN IRELAND
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