TOEET Vector Backbone for Recombinant Protein Expression

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Solution Overview

Problem

Current E. coli expression systems face challenges in producing recombinant proteins from higher eukaryotes, such as humans, due to low expression levels and misfolding, particularly due to mRNA secondary structure and codon bias issues, making high-level production costly and inefficient.

Innovation Solution

Development of Transcript-Optimized Expression Enhancement Technology (TOEET) that modifies the 5' untranslated region and adjacent polypeptide tag in expression vectors to minimize RNA secondary structure and optimize codon usage, allowing for high-level production of recombinant proteins without individual RNA optimization for each target gene.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If standard E. coli expression systems are used for higher eukaryotic proteins, then production cost is reduced, but expression level becomes insufficient

Engineering Contradiction:

Improveexpression levelVSAvoidproduction complexity

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent modifies nucleotide parameters in the 5' UTR and coding regions to optimize mRNA secondary structure and codon usage, thereby enhancing translation efficiency and protein expression levels without changing the host system

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent performs preliminary optimization of the expression vector backbone including RBS and 5' UTR sequences before target gene insertion, creating a pre-optimized system that eliminates the need for individual gene optimization later

Inventive Principle:

Principle #10Preliminary action

2Productivity

If individual RNA optimization is performed for each target gene, then expression level increases, but time and cost increase

Engineering Contradiction:

Improveexpression levelVSAvoidoptimization time

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The patent creates a universal optimized expression vector backbone that can be used for multiple different target genes, eliminating the need for individual optimization of each gene while maintaining high expression levels across diverse protein targets

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The optimization work is performed once on the expression vector backbone before use, rather than repeatedly for each target gene, significantly reducing the time and resources required for subsequent protein production projects

Inventive Principle:

Principle #10Preliminary action

3Productivity

If total gene synthesis is used for each protein target, then RNA optimization is achieved, but cost increases

Engineering Contradiction:

Improveexpression levelVSAvoidproduction cost

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The patent divides the optimization task into two segments: optimizing the expression vector backbone once, and using that optimized backbone for multiple target genes through simple cloning, rather than synthesizing optimized genes for each target

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent creates a master optimized expression vector that can be copied and used for multiple different target genes, avoiding the need for expensive total gene synthesis for each protein while maintaining all optimization benefits

Inventive Principle:

Principle #26Copying

Data Source

PatentUS10385350B2Transcript optimized expression enhancement for high-level production of proteins and protein domains

Publication Date: 2019.08.20 RUTGERS THE STATE UNIV

AI summary

The present invention relates to a system for high-level production of recombinant proteins and protein domains.