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High level expression of recombinant CRM197

A technology of CRM197 and expression vector, applied in the field of high-level expression of recombinant CRM197

Active Publication Date: 2015-06-24
PHOENIX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Methods for high-level expression of CRM197 for approved therapeutic and research use have not been reported

Method used

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  • High level expression of recombinant CRM197
  • High level expression of recombinant CRM197
  • High level expression of recombinant CRM197

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: High-throughput expression of recombinant CRM197 protein

[0113] A CRM197 expression strain was constructed, and the amount of soluble CRM197 protein produced in the strain was analyzed by capillary gel electrophoresis (SDS-CGE). Based on the data obtained, specific strains were selected for large-scale expression.

[0114] Construction and growth of CRM197 expression strain

[0115] The CRM 197 coding sequence was constructed using the preferential codons of Pseudomonas fluorescens to encode the CRM 197 amino acid sequence. figure 1 The amino acid and DNA sequences of the expressed synthetic CRM 197 gene are shown.

[0116] A standard set of secretion leaders and host strains were used. A plasmid carrying the codon-optimized CRM197 sequence fused to the 10 Pseudomonas fluorescens secretion leaders as shown in Table 6 was constructed. A leader sequence was included to target the protein to the periplasm where it is recovered in a properly folded and activ...

Embodiment 2

[0133] Example 2: Large-scale expression of recombinant CRM197 protein

[0134] Pseudomonas fluorescens Pfēnex Expression Technology TM Strains CS538-742, CS538-743, CS538-746, CS538-748, CS538-752 produced recombinant CRM197 protein in 2 liter fermentors.

[0135] Cultures were grown in 2 liter fermentors containing mineral salt medium as described herein and also in, eg, Riesenberg, D. et al., 1991, maintained at 32°C and pH 6.5 by addition of ammonium salts. Dissolved oxygen was maintained in excess by increasing agitation and sparging air and oxygen flow into the fermenter. Glycerol was delivered to the culture throughout the fermentation to maintain excess levels. These conditions were maintained until reaching the target cultured cell density for induction (absorbance at 575 nm (A 575 )), at which point IPTG is added to initiate target protein production. IPTG was added at a concentration of 0.5 mM to initiate CRM197 production. After 16-24 hours, the cultures from e...

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Abstract

The present invention relates to the field of recombinant protein production in bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of a recombinant CRM197 protein from a bacterial host.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Patent Application Serial No. 61 / 319,152, filed March 30, 2010, entitled "High Level Expression of Recombinant CRM197." Background of the invention [0003] Diphtheria toxin (DT) is a protein toxin synthesized and secreted by toxigenic strains of Corynebacterium diphtheriae. Toxigenic strains contain phage lysogens carrying toxin genes. DT is synthesized as a 535 amino acid polypeptide that undergoes proteolysis to form the mature toxin. The mature toxin contains two subunits, A and B, linked by a disulfide bond. The B subunit formed from the C-terminal portion of the intact DT enables DT to bind and pass through the cell membrane into the cytoplasm. Once inside the cell, the A subunit of the enzyme formed from the N-terminal portion of the intact DT catalyzes the ADP ribosylation of elongation factor 2 (EF-2). As a result, EF-2 is inactivated, protein synthesis stops and cells ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/78
CPCC07K14/34C12N1/20C12N15/78C12R2001/39Y10S435/876
Inventor D·M·雷塔拉克L·周H·金
Owner PHOENIX CORP