Protein Secretion Signals and Chaperones for High-Yield Expression
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Solution Overview
Problem
Existing host cells are not capable of efficiently producing and secreting proteins such as bovine lactoferrin, bovine lactoglobulin, or ovalbumin at high levels.
Innovation Solution
The use of genetically modified host cells with novel secretion signals and polynucleotides encoding CRT and PDIA3 proteins to enhance protein production and secretion, specifically incorporating secretion signals with defined amino acid sequences and overexpressing CRT, PDIA3, and optionally HAC1 proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional host cells are used for protein production, then the expression system is simple and easy to maintain, but the protein production and secretion levels are low
Solution Approach 1:
The secretion signal is divided into two functional segments: an N-terminal pre-sequence (15-30 amino acids) and a C-terminal pro-sequence (40-80 amino acids). This segmentation allows each region to perform its specific function optimally - the pre-sequence for targeting and the pro-sequence for folding and secretion - thereby improving protein secretion levels without overwhelming the expression system with unmanaged complexity.
Solution Approach 2:
The pre-sequence is designed to perform preliminary actions by directing the nascent protein chain to the endoplasmic reticulum membrane during translation. This preliminary targeting action occurs co-translationally, enabling the protein to be properly routed before full synthesis is complete, thus improving secretion efficiency while maintaining systematic organization.
2Productivity
If secretion signals with optimized amino acid sequences are used, then protein secretion efficiency is enhanced, but the design and implementation complexity increases
Solution Approach 1:
Specific amino acid parameters in the secretion signal are optimized within defined ranges: the pre-sequence is designed with 15-30 amino acids containing specific hydrophobic residues at key positions, while the pro-sequence comprises 40-80 amino acids with specific disulfide bond-forming cysteine pairs. These parameter optimizations enhance secretion efficiency while providing clear design guidelines that reduce implementation complexity through standardized specifications.
3Productivity
If host cells are genetically modified to overexpress CRT and PDIA3 proteins, then protein folding and secretion are improved, but the genetic modification process becomes more complex
Solution Approach 1:
CRT (calreticulin) and PDIA3 (protein disulfide isomerase A3) are introduced as intermediary proteins that mediate the folding and quality control of secreted proteins within the endoplasmic reticulum. These intermediaries assist the host cell's native folding machinery, improving overall secretion capacity without requiring fundamental changes to the host genome. The genetic modifications are limited to introducing these specific auxiliary proteins rather than reengineering the entire secretion pathway.
Data Source
AI summary
This disclosure provides expression systems comprising secretion signals that promote production and/or secretion of proteins of interest, as well as one or more polynucleotides encoding chaperone proteins (e.g., CRT and/or PDIA3) which, as demonstrate herein, enhance production and/or secretion of proteins. Moreover, genetically modified host cells comprising these expression systems are capable of producing high levels of protein of interest, such as bovine lactoferrin (bLF), bovine lactoglobulin (bLG), or ovalbumin (Ova).