Recombinant BoNT/E Production via Codon Optimization and Trypsin Activation
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Solution Overview
Problem
Current methods for producing recombinant Botulinum neurotoxin E (BoNT/E) are inefficient, requiring modification of the native cleavage site and resulting in low yields and purification challenges due to the need for non-native cleavage sites and inefficient in vitro oxidation of individual chains.
Innovation Solution
Designing nucleic acid sequences with optimized codon usage to enhance expression levels in E. coli, reducing the frequency of slow codons and using trypsin for activation to produce soluble di-chain BoNT/E without the need for exogenous cleavage sites, and employing hydrophobic purification to separate activated di-chain from trypsin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional C. botulinum culture methods are used for BoNT production, then the toxin can be produced in its native form, but the production efficiency is low and requires specialist culture equipment
Solution Approach 1:
The patent uses recombinant DNA technology to copy the BoNT/E gene into E. coli expression systems, replacing the need for C. botulinum culture. The nucleic acid sequences encoding BoNT/E are inserted into expression vectors that can be propagated in standard E. coli cultures, eliminating the need for specialist clostridial culture equipment while maintaining toxin production capability
Solution Approach 2:
The patent optimizes codon usage in the recombinant BoNT/E gene to match E. coli preferences, and modifies the expression system parameters (induction conditions, temperature, media composition) to maximize toxin yield. These parameter optimizations enable high-level expression in standard bacterial hosts without requiring native C. botulinum culture conditions
2Ease of manufacture
If non-native cleavage sites are inserted to enable activation, then the protein can be expressed in E. coli, but the purification becomes more difficult and yields are reduced
Solution Approach 1:
The patent removes the problematic non-native cleavage site modifications from the BoNT/E sequence. Instead, it uses the native cleavage site and employs controlled in vitro proteolytic activation after purification, separating the expression step from the activation step. This eliminates the purification difficulties associated with non-native cleavage sites while maintaining the ability to produce active toxin
Solution Approach 2:
The patent performs the proteolytic activation step as a preliminary action after purification but before final formulation. By expressing the full-length single-chain precursor without non-native cleavage sites, purifying the intact protein, and then activating it with specific proteases under controlled conditions, the method achieves both high expression levels and high purification yields
3Reliability
If in vitro oxidation of individual chains is used to form di-chain structure, then the active toxin can be produced, but the process is inefficient and yields are low
Solution Approach 1:
The patent merges the heavy and light chains into a single continuous polypeptide chain for expression, eliminating the need for separate oxidation steps. The single-chain precursor is expressed as one intact protein, purified in its reduced form, and then activated by proteolytic cleavage to generate the functional di-chain structure with native disulfide bonds, achieving both high reliability and high efficiency
4Productivity
If recombinant BoNT/E is produced with optimized codon usage, then expression levels are enhanced, but the sequence differs from native sequence
Solution Approach 1:
The patent applies codon optimization only to regions where it will not affect the protein sequence or function. The coding sequence is optimized for E. coli expression preferences while maintaining the exact amino acid sequence of native BoNT/E. The optimization focuses on synonymous codon substitutions that improve translation efficiency without altering the protein product, thus achieving both high expression levels and sequence fidelity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach results in high yields of soluble and active di-chain BoNT/E with improved solubility and reduced trypsin contamination, overcoming the limitations of existing methods by achieving efficient and pure protein production.
Implementation Method 1
contacting said single-chain solubilised BoNT/E with trypsin to produce activated di-chain BoNT/E
Implementation Method 2
employing hydrophobic purification to separate activated di-chain from trypsin
Data Source
Figure 1A
Figure 1B
Figure 2A
AI summary
The invention provides a nucleic acid sequence comprising a sequence of contiguous nucleotides, wherein said sequence of contiguous nucleotides has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 1, and wherein said sequence of contiguous nucleotides encodes a single-chain BoNT/E1 protein. The present invention also provides methods for producing soluble single-chain BoNT/E protein in an E. coli host cell, together with methods for producing soluble di-chain BoNT/E1 protein.