Codon optimized polynucleotide for high level expression of CRM197
Inventors
Goel, Akshay • Mishra, Ravi Pratap Narayan • Mantena, Narender Dev • Datla, Mahima
Assignees
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Publication Number
US-10280409-B2
Publication Date
2019-05-07
Expiration Date
Abstract
The present invention relates to high level expression of bacterial toxoid or toxin protein of pharmacological interest by means of an optimized novel polynucleotide sequence and host transformed with the said polynucleotide. Specifically, the invention provides a method for high production of polypeptide CRM197 wherein, the polynucleotide of the invention is used to transform a suitable host resulting in over-expression of corresponding proteins and a method for isolating the expressed polypeptide. More particularly, the present invention relates to high level expression of CRM197 in Escherichia coli and a method for the isolation and purification thereof.
Core Innovation
The invention relates to optimized CRM197 polynucleotides and polynucleotide variants, including a polynucleotide having the sequence set forth as SEQ ID NO. 2 and variants at least 85% homologous to SEQ ID NO. 2. Further polynucleotides are selected from SEQ ID NOs 3-10, and these polynucleotides are characterized for identity and structural equivalence to CRM197, including correct disulfide bonds and antigenic similarity.
The invention further provides expression constructs using the selected polynucleotide(s) to transform an Escherichia coli host cell, enabling high-level expression of a CRM197 polypeptide. The disclosed expression approach is described as induction-temperature-tunable, with an induction temperature range of 10-40°C used to influence soluble versus insoluble CRM197.
The invention also encompasses recovery of CRM197 polypeptide followed by purification, and characterization of the expressed CRM197. Characterization described includes SDS-PAGE, Western blot, SEC-HPLC, mass fingerprinting with 100% sequence similarity, N-terminal Edman degradation, CD/DSF secondary-structure similarity, and assessment that correct disulfide bonds are present, along with CRM197-specific ELISA demonstrating antigenic similarity.
The invention additionally states CRM197 as a carrier for conjugate vaccines, including conjugation with polysaccharides from multiple pathogens. The described document includes polysaccharides from Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, and Streptococcus pneumoniae, and describes conjugation with polysaccharide molecules.
Claims Coverage
The claims include five inventive features covering CRM197 polynucleotide sequences, expression in Escherichia coli, selected plasmid vectors, induction temperature control, and conjugation with pathogen polysaccharides.
Polynucleotide set defined by SEQ ID NO. 2 homology
A polynucleotide having the sequence set forth as SEQ ID NO. 2 or a variant thereof which is at least 85% homologous to SEQ ID NO. 2.
Polynucleotide variant set selected from SEQ ID NOs 3-10
A polynucleotide having the sequence selected from the group consisting of SEQ ID NO. 3, 4, 5, 6, 7, 8, 9, and 10.
Production of CRM197 polypeptide in Escherichia coli with induction temperature 10 to 40°C
A process producing the CRM 197 polypeptide by selecting a polynucleotide, inserting it into an Escherichia coli host cell for high level expression, maintaining an induction temperature of 10 to 40°C, and then extracting and purifying the CRM 197 polypeptide.
Expression vector plasmid options for transforming an Escherichia coli host
An expression vector configured to transform an Escherichia coli host cell, wherein the expression vector is a plasmid vector selected from pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pTWIN1, pTWIN2, pET12b, pET12c, and pET17b.
Conjugation of CRM197 polypeptide with pathogen polysaccharides
A process in which the CRM197 polypeptide is conjugated with polysaccharide molecules isolated from Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae, or other pathogenetic bacteria.
Overall claim coverage centers on CRM197 polynucleotide sequences and selected variants, including defined sequence sets (SEQ ID NO. 2 and SEQ ID NOs 3-10) and homology thresholds, together with expression in Escherichia coli using selected plasmid vector options. The claims also cover a production workflow with induction temperature 10-40°C and downstream conjugation of CRM197 with polysaccharides from enumerated pathogens.
Stated Advantages
High level expression of a CRM 197 polypeptide.
Purified CRM197 polypeptide characterized as having identity/structural equivalence, including correct disulfide bonds.
Antigenic similarity of the expressed CRM197 polypeptide shown by CRM197-specific ELISA.
CRM197 used as a carrier for conjugate vaccines via conjugation with polysaccharides from multiple pathogens.
Documented Applications
Conjugate vaccines in which CRM197 is used as a carrier and is conjugated with polysaccharides from Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, and Streptococcus pneumoniae.
Conjugation with polysaccharide molecules isolated from other pathogenetic bacteria.
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