PDI1 Fusion Vector for S. pombe Protein Secretion
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Solution Overview
Problem
Current methods for secretory protein production in S. pombe often result in insufficient yields and inefficient protein secretion, with co-expression systems facing issues like protein aggregation and incomplete passage through the secretory pathway.
Innovation Solution
An expression vector is developed that includes a structural gene sequence for a fusion protein containing a partial protein of PDI1, which serves as a molecular chaperone and ER targeting signal, along with a cleavage site for protease recognition, to enhance secretion efficiency by promoting proper folding and secretion of the protein of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a secretion signal peptide is added to the N-terminal of a foreign protein to enable secretory production, then the protein can be secreted from the host cell, but the production amount may not be sufficient depending on the protein type
Solution Approach 1:
The patent introduces PDI1 (protein disulfide isomerase 1) as an intermediary protein that facilitates the secretory pathway. PDI1 acts as a molecular chaperone that assists foreign proteins in passing through the secretory pathway, resolving the contradiction between achieving sufficient production amounts and maintaining reliable secretion efficiency for various protein types
Solution Approach 2:
The patent creates a fusion protein combining the foreign protein of interest with PDI1. This composite fusion protein leverages the molecular chaperone function of PDI1 to enhance the secretory production efficiency of the foreign protein, thereby increasing both production amount and secretion reliability simultaneously
2Productivity
If co-expression of a foreign secretory protein with PDI1 is performed to increase secretory production amount, then production efficiency improves, but protein coagulation occurs in the secretory pathway
Solution Approach 1:
The patent applies local quality by creating a fusion protein where PDI1 is expressed specifically in the context of the foreign protein's secretory pathway traversal. The fusion construct localizes the molecular chaperone function precisely where needed - during the secretory transit - without causing widespread coagulation throughout the cell
Solution Approach 2:
In the fusion protein construct, PDI1 serves as an intermediary molecular chaperone that specifically assists the foreign protein through the secretory pathway. This localized intermediary action prevents coagulation by providing targeted chaperone assistance rather than uncontrolled PDI1 expression
3Productivity
If co-expression system is used to increase secretory production, then production amount increases, but proteins do not pass through the secretory pathway efficiently
Solution Approach 1:
The patent merges the foreign protein with PDI1 in a fusion construct, combining the production function of the foreign protein with the secretory pathway facilitation function of PDI1. This merging ensures that the protein of interest is guided through the secretory pathway efficiently while maintaining high production amounts
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly increases the secretory production efficiency of proteins, allowing for high-yield recovery of the protein of interest from the culture broth by facilitating correct processing and secretion, even for proteins that may not efficiently pass through the secretory pathway.
Implementation Method 1
PDI1 (Protein disulfide isomerase 1) has a molecular chaperone function, and is a protein localized to the ER
Implementation Method 2
When cultivating a S. pombe transformant introduced with a structural gene encoding a fusion protein having the polypeptide at the N-terminal of a foreign protein, the produced fusion protein is separated into a signal peptide and the foreign protein in the Golgi apparatus or the endoplasmic reticulum (ER), and accordingly, the foreign protein will be secreted from the host cell into a culture broth
Implementation Method 3
a structural gene sequence (w), in between the structural gene sequence (y) and the structural gene sequence (z), encoding a cleavage site (W) that is comprised of an amino acid or a peptide and functions as a site to be cleaved at the N-terminal side of the protein (Z) moiety either in inside or outside of a cell
Data Source
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AI summary
Provided are: an expression vector for secreting a protein (Z) to be recovered or a fusion protein having the protein (Z) moiety therein; a method for producing a transformant using the expression vector; the transformant; and a method for producing a protein using the transformant. An expression vector comprising an expression cassette containing a structural gene sequence (y) encoding a protein (Y), a structural gene sequence (z) located downstream from the structural gene sequence (y) and encoding a protein (Z) that is a protein to be recovered, and a promoter sequence and a terminator sequence for expressing a fusion protein containing the protein (Y) moiety and the protein (Z) moiety, characterized in that the protein (Y) is a full-length protein of protein disulfide isomerase 1 (PDI1), a partial protein of PDI1, or a mutant protein of the full-length protein or the partial protein.