Heme-containing polypeptide secretion via TAT pathway
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Heme-containing polypeptides are difficult to secrete and fold properly in recombinant bacterial cells like Bacillus species due to the need for a non-covalently bound heme group to be inserted and maintained throughout the secretion process, with existing methods unclear on their expression and secretion.
Innovation Solution
A recombinant bacterium or plant cell is engineered with exogenous nucleic acid encoding a signal peptide and a heme-containing polypeptide, allowing for the secretion of the heme-containing polypeptide, where the signal peptide is removed upon secretion, and the heme group remains associated, using specific sequences and tags for efficient production and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If heme-containing polypeptides are secreted using the SEC pathway, then the protein can be secreted through the cell membrane, but the protein is unfolded during the process and cannot maintain the native heme configuration
Solution Approach 1:
The patent uses a signal peptide as an intermediary component that directs the heme-containing polypeptide through the TAT pathway instead of the SEC pathway. The signal peptide acts as a mediator that interacts with the transport system to ensure the polypeptide maintains its folded state with native heme configuration during secretion.
Solution Approach 2:
The patent changes the secretion pathway parameter from SEC to TAT by introducing a specific signal peptide sequence. This parameter change allows the polypeptide to be secreted in its folded state rather than unfolded, thereby maintaining the native heme group configuration throughout the secretion process.
2Stability of the object's composition
If heme-containing polypeptides are secreted using the TAT system, then the proteins can be secreted in the folded state, but it is unclear whether recombinant hemoproteins with non-covalently bound heme groups can be properly expressed and secreted
Solution Approach 1:
The patent applies preliminary action by first expressing the heme-containing polypeptide within the bacterial cell to ensure proper folding and heme binding occurs before secretion. The signal peptide is designed to facilitate subsequent secretion only after the polypeptide has achieved its native folded state with heme group properly bound, thereby ensuring both folded state maintenance and reliable secretion.
3Ease of operation
If signal peptide is used to direct secretion, then the heme-containing polypeptide can be secreted, but the signal peptide must be removed from the final product
Solution Approach 1:
The patent applies the extraction principle by designing the signal peptide to be cleaved off from the heme-containing polypeptide during or after secretion. The signal peptide serves its directional function during secretion and is then removed (taken out) from the final product, leaving only the pure heme-containing polypeptide without the signal peptide sequence.
Data Source
AI summary
This disclosure provides for methods and compositions for the expression and secretion of heme-containing polypeptides.