Mycobacteria-Escherichia coli Shuttle Vector Protein Folding

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Solution Overview

Problem

Current Mycobacteria-Escherichia coli shuttle vectors fail to correctly fold and modify proteins expressed in Mycobacteria, leading to inefficiencies in protein production.

Innovation Solution

Development of a novel Mycobacteria-Escherichia coli shuttle vector with a specific origin of replication (oriM) and promoter combinations, including A+T rich regions and direct repeat regions, to enhance protein expression and stability, using various promoters like heat shock protein, CMV, and ribosome RNA promoters, and encoding for target materials such as fluorescent proteins or therapeutic proteins.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the pAL5000 replicon is used as a Mycobacteria-Escherichia coli shuttle plasmid, then the plasmid can be replicated in both Mycobacteria and E. coli, but the protein expressed from the vector in mycobacteria is not correctly folded and modified

Engineering Contradiction:

Improvehost rangeVSAvoidprotein folding and modification

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent introduces specific Mycobacterial elements (oriM origin of replication and Mycobacterial promoters) into the shuttle plasmid structure to create a specialized replication and expression system that maintains compatibility with both E. coli and Mycobacteria while ensuring proper protein folding and modification in the Mycobacterial host

Inventive Principle:

Principle #3Local quality

2Ease of operation

If conventional shuttle vectors are used for protein expression in Mycobacteria, then the expression system is simple and easy to operate, but the protein expression levels are low and proteins are not correctly folded

Engineering Contradiction:

Improvevector system simplicityVSAvoidprotein expression level

Core Design Contradiction:

Ease of operationVSProductivity

Solution Approach 1:

The patent changes key parameters of the vector system by incorporating oriM origin of replication and Mycobacterial promoters to optimize protein expression levels and folding accuracy while maintaining the dual-host replication capability and operational simplicity

Inventive Principle:

Principle #35Parameter changes

3Reliability

If the pAL5000 replicon is used, then the vector system is well-established and widely used, but the system cannot reliably produce correctly folded and modified proteins

Engineering Contradiction:

Improvesystem reliabilityVSAvoidprotein folding accuracy

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent creates a composite vector system that integrates elements from both E. coli and Mycobacteria (including oriM origin, Mycobacterial promoters, and selection markers) to achieve reliable dual-host replication while ensuring accurate protein folding and modification through Mycobacterial-specific regulatory elements

Inventive Principle:

Principle #40Composite materials

Data Source

PatentUS8841432B2Shuttle vectors for mycobacteria-escherichia coli and uses thereof

Publication Date: 2014.09.23 SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION

AI summary

The present disclosure provides a DNA molecule capable of replication in Mycobacteria having a nucleic acid sequence as disclosed in SEQ ID NO: 1, a shuttle vector constructed using it and a transformed cells containing the present vector. The vector of about 18 kb of the present disclosure contains 16 ORFs, a replication origin and a rep-like protein essential for replication. Therefore, the plasmid of the present disclosure can be utilized as a gene delivery system/research, and also in a therapeutic system such as immune therapeutics by effectively delivering proteins or heterologous DNA and expressing the encoded DNA in cells.