Recombinant Host Cells Using Multiple Secretion Signals
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Solution Overview
Problem
Current methods for producing recombinant proteins in recombinant host cells face limitations in achieving high secreted yields due to genetic instability and saturation of the secretory pathways, leading to accumulation of unfolded or mis-folded proteins and activation of molecular stress responses.
Innovation Solution
The use of recombinant host cells that comprise multiple polynucleotide sequences encoding a recombinant protein operably linked to at least two distinct secretion signals, which allows for engagement of distinct cellular secretory pathways and prevents over-saturation of any one pathway.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If high copy numbers of polynucleotide sequences are engineered into recombinant host cells to increase production, then the quantity of recombinant protein increases, but genetic stability deteriorates and yields plateau or decline
Solution Approach 1:
The invention divides the production burden by using multiple distinct secretion signals (e.g., alpha-factor signal peptide, PEP4 signal peptide, DSE4 signal peptide) instead of relying on high copy numbers of a single polynucleotide sequence. Each signal directs the recombinant protein through different secretory pathways, segmenting the overload problem into manageable channels and maintaining genetic stability while achieving high yields.
2Quantity of substance
If strong promoters are used to increase transcription of polynucleotide sequences, then the quantity of recombinant protein increases, but the secretory pathways become saturated and yields plateau
Solution Approach 1:
The invention segments the secretory load by employing multiple distinct secretion signals that direct proteins through different cellular pathways. This prevents saturation of any single secretory pathway while maintaining high production levels, thereby preserving secretion efficiency despite strong promoter activity.
Solution Approach 2:
The invention changes the parameter of secretion signal diversity by using at least two distinct signals with different cellular recognition patterns. This parameter change allows the system to handle high protein loads without pathway saturation, maintaining productivity even when strong promoters drive high transcription.
3Quantity of substance
If multiple distinct secretion signals are used to direct recombinant protein secretion, then secreted yields increase and pathway saturation is prevented, but device complexity increases
Solution Approach 1:
The invention applies multi-functionality by designing a recombinant vector framework that can accommodate multiple different secretion signals (alpha-factor, PEP4, DSE4, EPX1, CLSP) within a single construct. This universal platform handles diverse secretion pathways simultaneously, achieving high secreted yields without proportionally increasing operational complexity.
Data Source
AI summary
The present disclosure relates to methods for producing recombinant proteins, as well as compositions used in and produced by such methods. Specifically, the present disclosure relates to methods for producing high secreted yields of recombinant proteins, and the compositions provided herein include recombinant host cells that comprise polynucleotide sequences encoding proteins operably linked to at least 2 distinct secretion signals.