Fusion Polypeptide Cleavage Site Engineering for Higher Purity
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Solution Overview
Problem
Existing fusion polypeptide production methods often result in incomplete cleavage, leading to suboptimal yields of the polypeptides of interest due to inefficient protease-mediated cleavage.
Innovation Solution
Engineered fusion polypeptides with altered amino acid sequences in the protease recognition site pre- and/or post-sequences, specifically incorporating substitutions such as hydrophobic, aromatic, and basic amino acids, enhance protease-mediated cleavage, particularly by KEX2 serine peptidase, improving secretion and purification of polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional fusion polypeptide sequences are used, then production is simpler, but cleavage is incomplete leading to suboptimal yields
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence parameters at the protease recognition site. Specifically, it optimizes the pre-sequence and post-sequence flanking the cleavage site by selecting specific amino acids (e.g., basic residues like Arginine or Lysine at positions -3 and -6, and specific residues at positions -1, +1, +2) to enhance protease recognition and cleavage efficiency, thereby resolving the contradiction between productivity and reliability of cleavage.
Solution Approach 2:
The patent applies local quality by focusing modifications specifically at the protease recognition site rather than the entire fusion polypeptide sequence. By optimizing only the critical pre-sequence and post-sequence regions that flank the cleavage site, the invention achieves improved cleavage reliability without unnecessarily complicating the overall production system.
2Manufacturing precision
If amino acid substitutions are introduced to improve cleavage, then purification efficiency increases, but sequence complexity increases
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions only at specific positions in the pre-sequence and post-sequence flanking the protease recognition site, rather than modifying the entire fusion polypeptide sequence. This targeted approach improves manufacturing precision (purification efficiency) while minimizing sequence complexity increases.
Solution Approach 2:
The patent applies parameter changes by systematically optimizing specific amino acid positions (e.g., positions -6, -3, -1, +1, +2 relative to the cleavage site) with defined residue preferences. This structured parameter optimization achieves high purification efficiency while maintaining manageable sequence complexity through rational design rather than random modification.
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
The engineered fusion polypeptides exhibit enhanced secretion and purification of polypeptides, allowing for quicker scale-up and higher purity of therapeutic proteins like antibodies with reduced contamination by uncleaved products.
Implementation Method 1
protease-mediated (such as, KEX2-mediated) cleavage
Implementation Method 2
KEX2 serine peptidase
Data Source
AI summary
Provided herein, inter alia, are fusion DNA constructs comprising improved protease recognition sequences for expressing and purifying one or more polypeptides of interest as well as methods for producing one or more polypeptides of interest in a recombinant host cell.