Yeast Secretion Signals for High-Yield Recombinant Protein Production
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Solution Overview
Problem
Existing methods for producing recombinant proteins in yeast cells face limitations in achieving high secreted yields due to genetic destabilization and misfolding, leading to accumulation of unfolded proteins and activation of stress responses, which bottleneck the secretion machinery.
Innovation Solution
Employing recombinant secretion signals comprising a functional variant of the leader peptide of the α-mating factor of Saccharomyces cerevisiae (*pro-αMF (sc)) in combination with specific signal peptides to enhance the secretion of recombinant proteins, thereby alleviating the negative impact on host cells and increasing yields.
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 recombinant protein production, then protein expression level is improved, but genetic stability deteriorates due to cellular genetic destabilization
Solution Approach 1:
The patent changes the parameter of polynucleotide copy number from high copy numbers (2 or more copies) to low copy numbers (0.1 to 1 copy per host cell genome). This parameter change resolves the contradiction by maintaining genetic stability while achieving sufficient protein production through optimization of other parameters such as promoter strength and secretion signal efficiency
Solution Approach 2:
The patent uses multiple copies of the same polynucleotide sequence in different host cells rather than multiple copies within a single cell. This approach maintains genetic stability in each individual cell while achieving high overall protein production through cultivation of multiple host cells, each contributing to the total yield
2Quantity of substance
If strong promoters are used to increase transcription of polynucleotide sequences and thereby increase recombinant protein expression, then protein expression level is improved, but cellular stress response is activated leading to misfolding and accumulation of unfolded proteins
Solution Approach 1:
The patent changes the parameter of promoter strength from strong promoters to moderate promoters. This parameter change allows sufficient protein expression while avoiding activation of cellular stress responses that would otherwise occur with overly strong promoters, thereby preventing misfolding and accumulation of unfolded proteins
Solution Approach 2:
The patent introduces secretion signals as intermediary elements that facilitate the export of recombinant proteins from the host cell. This intermediary mechanism reduces the burden on the cellular folding and secretion machinery, allowing high protein expression without activating stress responses or causing misfolding
3Productivity
If high density cell growth is achieved in yeast cells to increase recombinant protein production, then productivity is improved, but secretion machinery becomes saturated leading to accumulation of unfolded proteins
Solution Approach 1:
The patent changes the parameter of cell density from high density to low density cell growth conditions. This parameter change prevents saturation of the secretion machinery while maintaining sufficient productivity through optimization of other parameters such as protein secretion efficiency and host cell strain selection
Solution Approach 2:
The patent uses secretion signals as intermediary elements that enhance the efficiency of protein export from the cell. This intermediary mechanism increases the capacity of the secretion pathway, allowing high productivity without saturation or accumulation of unfolded proteins
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 expression constructs, recombinant vectors, and recombinant host cells that comprise polynucleotide sequences encoding proteins operably linked to recombinant secretion signals that comprise the leader peptide of the α-mating factor (αMF) of Saccharomyces cerevisiae and a non-αMF signal peptide.