Yeast Helper Factor Co-Expression for Higher Protein Secretion
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for producing heterologous proteins in yeast hosts face challenges in achieving optimal expression and secretion yields, as co-overexpression of chaperones can have varying effects, sometimes leading to diminished productivity or no additional benefit, and there is a need for improved host cells to enhance protein production.
Innovation Solution
A method involving the co-expression of three helper factors in yeast host cells, including a chaperone and two co-chaperones with high sequence identity to human proteins, such as hBiP, hGrp170, and hERdj3, encoded by specific nucleotide sequences optimized for yeast expression, using heterologous expression cassettes with secretion signals and ER retention sequences, to enhance protein secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple chaperones are co-overexpressed in yeast host cells, then protein folding and secretion may be enhanced, but productivity is often diminished or no additional benefit is achieved
Solution Approach 1:
The patent extracts and identifies specific limiting factors from the complex chaperone system (BiP/Kar2, NEFs Lhs1/Sil1, and J-proteins ERdj3/Scj1) that are responsible for bottlenecking protein secretion. By targeting only these specific factors for overexpression rather than all chaperones, the patent resolves the contradiction by removing the limiting elements without causing the overload effect that diminishes productivity.
Solution Approach 2:
The patent changes the expression levels of specific chaperone parameters (BiP/Kar2, Lhs1/Sil1, ERdj3/Scj1) from normal to overexpressed states. This parameter change optimizes the chaperone system's capacity to handle protein folding and secretion, thereby improving productivity while maintaining reliable folding and secretion functions.
2Quantity of substance
If chaperones are co-overexpressed to enhance protein production, then secretion may be improved in some cases, but sometimes it has a diminishing effect or negative effect on productivity
Solution Approach 1:
The patent introduces specific intermediary chaperone factors (BiP/Kar2, Lhs1/Sil1, ERdj3/Scj1) that mediate between protein synthesis and secretion processes. These intermediaries facilitate efficient protein folding and secretion without causing the negative effects associated with non-specific chaperone overexpression, thus improving both protein secretion and productivity.
3Reliability
If multiple folding factors are combined to improve antibody secretion, then folding and secretion may be enhanced, but the combination does not induce additional effect compared to just one element
Solution Approach 1:
The patent segments the chaperone system into distinct functional modules: BiP/Kar2 for general protein binding, Lhs1/Sil1 for nucleotide exchange, and ERdj3/Scj1 for J-protein chaperoning. By segmenting and targeting each module specifically rather than combining all possible factors, the patent achieves reliable folding and secretion with reduced system complexity.
Data Source
AI summary
A method for producing of a protein of interest (POI) in a yeast host cell that is modified to comprise within one or more expression cassettes heterologous nucleic acid molecules encoding helper factors and a gene of interest (GOI) encoding the POI, wherein: a) a first helper factor comprises at least 90% sequence identity to SEQ ID NO:1; b) a second helper factor comprises at east 90% sequence identity to SEQ ID NO:3; and c) a third helper factor comprises at least 90% sequence identity to SEQ ID NO:5; which method comprises (i) culturing said host cell in a culture medium under conditions to co-express said heterologous nucleic acid molecules and to secrete said POI into the host cell culture; and (ii) recovering the POI from the host cell culture.