Bacterial Expression Vector with Truncated YebF Carrier
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Solution Overview
Problem
Current methods for producing recombinant proteins in Escherichia coli face challenges such as incorrect protein export, aggregation in the cytoplasm, cell lysis, incorrect folding, and proteolytic degradation, which limit the volume and functionality of protein production.
Innovation Solution
A novel bacterial expression vector is developed, featuring a secretory signal sequence that combines DNA sequences of signal peptides from genes like pelB, ompA, yebF, and ompF with a DNA sequence encoding a truncated yebF carrier protein. This vector enhances the secretion of recombinant proteins into extracellular media, facilitating easy purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional secretory signal peptides are used for protein secretion in E. coli, then protein export is facilitated, but protein aggregation in cytoplasm and incorrect folding occur
Solution Approach 1:
The invention introduces a two-component secretory signal system consisting of a conventional signal peptide (pelB, ompA, yebF, or ompF) combined with a truncated yebF carrier protein. The signal peptide mediates initial translocation to the periplasm, while the truncated yebF protein acts as an intermediary carrier that prevents aggregation and facilitates proper folding in the periplasmic environment, thereby resolving the contradiction between secretion volume and protein functionality.
Solution Approach 2:
The secretory signal sequence is constructed as a composite of two functional elements: (1) DNA sequences encoding conventional signal peptides (pelB, ompA, yebF, or ompF) and (2) DNA sequences encoding a truncated yebF carrier protein. This composite structure combines the translocation capability of signal peptides with the protective and folding-assistance properties of the truncated yebF carrier, achieving both high secretion volume and maintained protein functionality.
2Productivity
If target cell adaptation is performed to enhance extracellular production, then secretion efficiency improves, but cell lysis and proteolytic degradation increase
Solution Approach 1:
The truncated yebF carrier protein serves as a protective intermediary that shields the recombinant protein from proteolytic degradation during the secretion process. It maintains the protein in a stable, folded state in the periplasm, preventing exposure to extracellular proteases and reducing cell lysis-related damage, thereby enabling high extracellular production without increased degradation.
Solution Approach 2:
The truncated yebF carrier protein provides beforehand cushioning protection to the recombinant protein during translocation and in the periplasmic space. This protective mechanism is in place before the protein encounters harsh extracellular conditions, preventing cell lysis and proteolytic degradation in advance, thus maintaining protein integrity during high-volume secretion.
3Ease of operation
If signal peptides are used for protein translocation to periplasm, then secretion is enhanced, but protein aggregation in cytoplasm occurs
Solution Approach 1:
The truncated yebF carrier protein acts as a mediating chaperone that accompanies the recombinant protein during translocation to the periplasm. It prevents cytoplasmic aggregation by maintaining the protein in a soluble, translocation-competent state, and continues to protect it in the periplasm, thereby ensuring both efficient translocation and prevention of aggregation.
Solution Approach 2:
The secretory signal sequence combines the translocation-functioning signal peptide with the aggregation-preventing truncated yebF carrier protein. This composite structure ensures that while the signal peptide drives efficient translocation to the periplasm, the truncated yebF carrier simultaneously prevents cytoplasmic aggregation and maintains protein stability throughout the secretion process.
Data Source
AI summary
The invention provides a bacterial expression vector (100) comprising of a secretory signal sequence in tandem with DNA sequence encoding recombinant protein (103), wherein, the secretory signal sequence is a combination comprising of: a) at least one DNA sequence encoding a signal sequence (101) of gene selected from the group consisting of pelB, ompA, yebF, and ompF, and b) at least one DNA sequence encoding a carrier peptide (102) selected from the group consisting of Seq. ID 5 and 6 encoding truncated yebF.