N-terminal Cleaved Soluble PH20 Polypeptide for Expression
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Solution Overview
Problem
Current methods for increasing the expression level of industrially useful proteins secreted from recombinant cells face challenges such as inefficient protein folding, imprecise signal peptide cleavage, and increased immunogenicity due to amino acid modifications.
Innovation Solution
The development of an N-terminal and/or C-terminal cleaved soluble PH20 polypeptide, which involves deleting 1 to 7 amino acid residues from the N-terminus of the mature animal wild-type PH20, to enhance productivity and maintain enzymatic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If signal peptides are substituted to increase protein expression, then expression level increases, but signal peptidase fails to cleave precisely between the signal peptide and the N-terminal amino acid of mature protein
Solution Approach 1:
The patent removes the problematic signal peptide sequence and replaces it with an alternative signal peptide that does not interfere with precise cleavage. This extraction of the harmful element (the original signal peptide that causes imprecise cleavage) while retaining the necessary function (signal for secretion) resolves the contradiction between increased expression and precise cleavage.
Solution Approach 2:
The patent modifies the amino acid sequence at the N-terminus by substituting specific residues (e.g., changing the sequence from MT... to a modified version) to create a variant that is both highly expressed and precisely cleaved. This parameter change in the protein sequence allows optimization of both expression level and cleavage precision simultaneously.
2Productivity
If amino acid substitution is performed to increase protein expression, then protein folding environment improves, but immunogenicity increases
Solution Approach 1:
The patent applies local quality modification by making targeted amino acid substitutions only at specific positions (e.g., positions 2-7 of the N-terminal sequence) rather than throughout the entire protein. This localized modification improves folding and expression while minimizing changes that would trigger immune responses in the bulk of the protein structure.
Solution Approach 2:
The patent systematically varies specific amino acid parameters (hydrophobicity, charge, size) at defined positions to optimize expression while maintaining immunological compatibility. By carefully selecting substitutions that improve folding kinetics without creating highly immunogenic epitopes, the patent resolves the contradiction between expression enhancement and immunogenicity reduction.
3Productivity
If N-terminal amino acid residues are deleted to improve productivity, then expression level and enzymatic activity increase, but protein structure may be affected
Solution Approach 1:
The patent extracts and removes the first 1-7 amino acid residues from the N-terminus of the PH20 polypeptide. This deletion of the terminal residues improves productivity by enhancing solubility and expression levels while the patent demonstrates that the core three-dimensional structure and catalytic function remain intact, resolving the contradiction between productivity and structural stability.
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 improves the productivity of soluble recombinant PH20 polypeptide, leading to higher expression levels and enzymatic activity, while minimizing immunogenicity concerns.
Implementation Method 1
Hyaluronidase is an enzyme that breaks down hyaluronan. Hyaluronidase can be used to treat diseases or disorders associated with accumulation of hyaluronan or other glycosaminoglycan by catalyzing hydrolysis of hyaluronan.
Data Source
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AI summary
The present invention relates to a recombinant PH20 polypeptide in which 1 to 7 amino acid residues are deleted at the N-terminus of a mature animal wild-type PH20, and use thereof. The N-terminal and/or C-terminal cleaved PH20 polypeptide with improved enzymatic activity and productivity presented in the present invention may exhibit superior expression and enzymatic activity compared to conventional recombinant PH20 polypeptides, which may result in reduced production costs for industrial use and thus reduced treatment costs.