Modifying Pichia pastoris Secretion Signals for Homogeneous IgV
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The production of immunoglobulin single variable domains in Pichia pastoris yeast strains often results in heterogeneous products due to incomplete processing of secretion signal sequences, leading to N-terminal extensions, which complicates purification and increases immunogenicity, thereby affecting the quality and cost of therapeutic applications.
Innovation Solution
Modifying the secretion signal sequence by substituting or deleting amino acid residues at non-canonical cleavage sites, such as those present in the native aMF secretion signal sequence, to reduce the presence of N-terminal extensions, ensuring less than 5% of the produced immunoglobulin single variable domains contain residual amino acids from the signal sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If the native aMF secretion signal sequence is used in Pichia pastoris, then secretion of immunoglobulin single variable domains is achieved, but incomplete processing leads to N-terminal extensions and product heterogeneity
Solution Approach 1:
The patent modifies the secretion signal sequence by changing specific amino acid residues (e.g., substituting residue 76 with R, H, A, or P; substituting residue 77 with T or A; or deleting residues 76-78) to optimize processing by Pichia pastoris enzymes. This parameter change in the signal sequence structure eliminates N-terminal extensions while preserving secretion capability, thereby resolving the contradiction between ease of secretion and product homogeneity.
2Productivity
If secretion signal sequences are used to direct protein secretion, then proteins are released into culture medium, but residual signal sequence amino acids remain attached to N-terminus
Solution Approach 1:
The patent systematically modifies the secretion signal sequence parameters by substituting or deleting specific amino acid residues at positions 76, 77, and 78. These parameter changes ensure complete cleavage by signal peptidase and absence of residual amino acids, achieving both high secretion efficiency and accurate amino acid sequence without N-terminal extensions.
3Ease of operation
If the alpha-factor signal sequence is used for secretion, then protein secretion is achieved, but Ste13 dipeptidyl aminopeptidase does not efficiently clip the signal peptide in Pichia pastoris
Solution Approach 1:
The patent identifies that the native alpha-factor signal sequence parameters are incompatible with Pichia pastoris Ste13 enzyme specificity. By changing specific amino acid parameters (substituting residue 76 with R/H/A/P, substituting residue 77 with T/A, or deleting residues 76-78), the signal sequence is optimized for complete processing by Pichia enzymes, ensuring reliable cleavage and elimination of N-terminal extensions while maintaining secretion function.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach significantly enhances the homogeneity of immunoglobulin single variable domains, reducing production costs and immunogenicity, and maintaining high yields of intact products, as demonstrated by mass spectrometry analysis.
Implementation Method 1
the secretion of the protein can be achieved by fusing the protein to a secretion signal sequence that directs the passage of the polypeptide through the endoplasmic reticulum and the Golgi apparatus and that is cleaved from the polypeptide by a signal peptidase
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Methods are provided for the expression of immunoglobulin variable domains that are secreted into the culture medium. The methods provide for the production of homogeneous immunoglobulin variable domains in which the proportion of product-related variants that comprise, at the N-terminus, at least one redundant amino acid residue derived from the secretion signal is strongly reduced or absent.