Yeast Expression of Native Human ER Chaperones
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Solution Overview
Problem
Current methods for producing recombinant human ER chaperones in microbial hosts, such as E. coli, lack the eukaryotic secretion pathway, leading to uncertainty about correct folding and functionality of the produced proteins, which are essential for therapeutic applications.
Innovation Solution
Utilizing native signal sequences of human ER chaperones in yeast expression systems like S. cerevisiae and P. pastoris to achieve correct processing and secretion of recombinant ER chaperones, ensuring they are processed similarly to native proteins and maintaining their biological activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant human ER chaperones are produced in bacterial host E. coli, then production efficiency and yield are improved, but correct folding and biological activity cannot be ensured due to lack of eukaryotic secretion pathway
Solution Approach 1:
The patent uses yeast (Pichia pastoris and Saccharomyces cerevisiae) as an intermediary host system that possesses both efficient recombinant protein production capabilities and complete eukaryotic secretion pathways. The yeast expression system acts as a mediator between bacterial high-yield production and the need for proper eukaryotic protein folding/processing, enabling production of correctly folded human ER chaperones with high yield and biological activity.
2Ease of operation
If conventional yeast protein secretion signal is fused to human ER protein sequence, then secretion to culture medium is achieved, but non-native amino acids are introduced on N-terminus affecting biological activity
Solution Approach 1:
The patent extracts and utilizes the native signal sequence that is already present in the human ER chaperone protein coding sequence. Instead of adding a foreign yeast secretion signal, the method removes unnecessary portions and retains only the functional native signal sequence, allowing the protein to be secreted to the culture medium while maintaining its native N-terminal structure and biological activity.
Solution Approach 2:
The patent enables the human ER chaperone protein to serve its own secretion function by utilizing its inherent native signal sequence. The protein's own sequence contains the necessary information for yeast to recognize and process it for secretion, eliminating the need for external modification with non-native secretion signals.
3Manufacturing precision
If human tissues are used as source for ER chaperone proteins, then native protein structure is obtained, but insufficient quantity is available for large scale clinical trials
Solution Approach 1:
The patent creates recombinant copies of human ER chaperone genes and expresses them in yeast hosts. This copying approach allows production of large quantities of proteins that are identical to native human proteins, overcoming the limitation of insufficient tissue availability while maintaining native structure and biological activity through proper eukaryotic expression systems.
Data Source
AI summary
A method using yeast as a host for production of human ER chaperone proteins, using endogenous signal peptides of intracellular human proteins that are recognized and correctly processed in the yeast cells to subsequently lead to the secretion of the human proteins. The resultant proteins possessed native amino acid sequence and were biologically active. Moreover, secretion allowed simple one-step purification of native recombinant human proteins with high yields.