A versatile bacterial expression vector based on the synthetic biology plasmid pSB1

Area of Science:

• Molecular Biology
• Synthetic Biology
• Protein Expression

Background:

• Escherichia coli is a widely used host for recombinant protein production.
• Development of versatile expression vectors is crucial for efficient protein engineering.
• Existing vectors may lack features for optimized fusion protein construction and targeted expression.

Purpose of the Study:

• To develop a novel Escherichia coli expression vector for streamlined construction and production of fusion proteins.
• To engineer a vector with enhanced features for soluble and active protein expression.
• To validate the vector's efficiency in expressing a target protein in different cellular compartments.

Main Methods:

• Design and assembly of a synthetic biology-based expression vector (pMD204) on the pSB1C3 platform.
• Incorporation of key elements: T7 promoter with lac operator, OmpA signal sequence, multiple cloning sites, and a C-terminal His-10 tag.
• Stepwise addition of three de novo designed cassettes, with flanking restriction sites for modularity.
• Expression of a cysteine proteinase inhibitor variant in the cytoplasm and periplasm of E. coli.

Main Results:

• Successful construction of the pMD204 expression vector with integrated functional elements.
• Demonstration of easy vector modification, including signal sequence removal without frame disruption.
• Production of a soluble and active cysteine proteinase inhibitor variant in both E. coli cytoplasm and periplasm.
• Confirmation of the vector's utility for producing functional fusion proteins.

Conclusions:

• The pMD204 vector provides a robust and flexible platform for fusion protein expression in Escherichia coli.
• The vector facilitates targeted protein localization and enhances the production of soluble, active proteins.
• pMD204 represents a valuable tool for synthetic biology and protein engineering applications.