Pichia pastoris 18-amino acid signal sequence for full-length protein secretion
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Solution Overview
Problem
Current signal sequences used in Pichia pastoris for heterologous protein expression, such as those with dibasic amino acid cleavage sites (KR and RR), face challenges in protein fragmentation due to internal Kex2p cleavage sites, limiting the secretion of full-length proteins.
Innovation Solution
A unique 18-amino acid signal sequence from Pichia pastoris is identified and utilized for heterologous protein expression, which is cleaved independently of Kex2p sites, allowing efficient secretion of proteins with internal dibasic amino acids into the medium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If signal sequences with dibasic amino acid cleavage sites (KR and RR) are used in Pichia pastoris for heterologous protein expression, then efficient secretion of proteins is achieved, but proteins with internal dibasic amino acids get fragmented due to Kex2p cleavage
Solution Approach 1:
The patent extracts and utilizes the native Pichia pastoris signal sequence from the DDDK protein, removing the dependency on Kex2p cleavage sites. This native signal sequence is naturally present in Pichia pastoris and directs secretion without requiring external Kex2p processing, thereby preventing fragmentation of proteins containing internal dibasic amino acids while maintaining efficient secretion
Solution Approach 2:
The patent introduces an alternative secretion pathway by using the native Pichia pastoris signal sequence as an intermediary mechanism. Instead of relying on the heterologous Kex2p system from Saccharomyces cerevisiae, the native signal sequence mediates protein secretion through Pichia's own secretory pathway, avoiding the harmful Kex2p cleavage action on internal dibasic sites
2Ease of manufacture
If Kex2p cleavage sites are used for signal sequence processing, then signal peptide removal is efficient, but proteins with internal Kex2p sites undergo unwanted cleavage and fragmentation
Solution Approach 1:
The patent extracts the signal sequence requirement from the Kex2p processing system and satisfies it using the native Pichia pastoris signal sequence. This extraction eliminates the need for Kex2p cleavage sites in the expression construct, allowing signal peptide removal through the native signal sequence while preserving protein integrity
Solution Approach 2:
Instead of using a heterologous signal sequence (from S. cerevisiae) that requires Kex2p processing, the patent inverts the approach by using the native Pichia pastoris signal sequence that operates independently of Kex2p. This inversion resolves the contradiction by making the signal processing system compatible with the host organism's native pathways
Data Source
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AI summary
The present invention relates to a signal sequence from a unique Pichia pastoris protein. Further the invention discloses use of signal sequence for the expression of heterologous protein in Pichia pastoris.