Co-expressing Ero1 and XBP1 to Boost Recombinant Protein Yield
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Solution Overview
Problem
Current methods for improving protein yield in expression systems, such as those involving the unfolded protein response (UPR) pathway, do not adequately increase the production capacity of recombinant proteins, particularly when relying on individual components like Ero1 or XBP1 alone.
Innovation Solution
Increasing the expression levels of both Ero1 and XBP1 in a recombinant host cell to enhance protein folding capacity and yield, achieved through the use of exogenous polynucleotide sequences encoding these proteins or modulation of endogenous sequences to up-regulate their expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If individual UPR components (Ero1 or XBP1) are expressed to enhance protein folding capacity, then protein folding capability is improved, but protein yield is insufficient
Solution Approach 1:
The patent combines expression of multiple UPR components (Ero1 and XBP1) into a unified expression system. By co-expressing these components that work together in the protein folding pathway, the system achieves synergistic enhancement of both folding capability and protein yield, resolving the contradiction between reliability and productivity.
2Reliability
If Ero1 expression is increased to enhance disulphide bond formation, then disulphide bond formation capability is improved, but overall protein production is limited
Solution Approach 1:
The patent merges Ero1 expression with XBP1 expression in a coordinated manner. Since XBP1 regulates the unfolded protein response and ER expansion, combining it with Ero1 creates a comprehensive enhancement of the protein folding system, thereby improving overall protein production while maintaining disulphide bond formation capability.
Solution Approach 2:
The patent changes the expression parameters by co-expressing multiple proteins (Ero1 and XBP1) rather than increasing the expression of a single component. This parameter change in the expression system allows simultaneous improvement of disulphide bond formation and overall protein production capacity.
3Reliability
If XBP1 expression is increased to up-regulate ER chaperone proteins and expand ER, then protein folding capacity is improved, but protein yield remains insufficient
Solution Approach 1:
The patent combines XBP1 expression with Ero1 expression to create a more comprehensive protein folding enhancement system. While XBP1 expands ER capacity and up-regulates chaperones, the addition of Ero1 specifically enhances disulphide bond formation, together achieving both improved folding capacity and increased protein yield.
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
The co-expression of Ero1 and XBP1 significantly improves protein yield, with specific productivity increasing by up to 800% or more compared to unmodified cells, and results in higher levels of correctly folded proteins, improved cell growth, and stability.
Implementation Method 1
Ero1 is a flavin adenine dinucleotide (FAD) dependent enzyme which facilitates disulphide bond formation in immunoglobulin subunits by selectively oxidizing PDI
Implementation Method 2
Proteins are oxidized by PDI when it is in an oxidized state, which produces a disulphide bond in the protein and renders PDI in a reduced state comprising free sulfhydryl groups
Data Source
AI summary
The present invention relates to a recombinant host cell, wherein the cell is modified to increase the expression levels of Ero1 and XBP1 relative to the expression levels of Ero1 and XBP1 in an unmodified cell. The present invention also relates to a method of producing a recombinant protein of interest comprising expressing the recombinant protein of interest in the recombinant host cell.