PH20 Hyaluronidase Variants for Thermal Stability and Expression
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Solution Overview
Problem
Current recombinant human PH20 hyaluronidase variants suffer from insufficient thermal stability and expression levels, limiting their effectiveness in clinical applications, particularly in cancer treatment.
Innovation Solution
Development of novel PH20 variants with specific amino acid residue substitutions in the alpha-helix and linker regions, along with selective deletions of N- and C-terminal residues, enhancing enzymatic activity and thermal stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current recombinant human PH20 hyaluronidase variants are used, then enzymatic activity is maintained, but thermal stability is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues at positions 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, and 363 in the PH20 hyaluronidase sequence. These amino acid substitutions are designed to improve thermal stability while preserving or enhancing enzymatic activity, directly resolving the technical contradiction between reliability and productivity.
2Productivity
If current recombinant human PH20 hyaluronidase variants are used, then enzymatic function is preserved, but expression levels are insufficient
Solution Approach 1:
The patent modifies amino acid parameters at specific positions (341-363) to optimize protein expression levels. The selected amino acid substitutions are intended to improve solubility, folding efficiency, and overall expression levels in recombinant systems while maintaining the catalytic function of the enzyme, thereby resolving the contradiction between productivity and reliability.
3Reliability
If amino acid substitutions are made in the alpha-helix region, then thermal stability is improved, but protein structure complexity increases
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions specifically in the alpha-helix region (positions 341-363) rather than throughout the entire protein structure. This localized modification approach improves thermal stability by stabilizing the alpha-helix structure while minimizing overall structural complexity changes, as only specific local regions are modified rather than the entire protein.
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 modified PH20 variants exhibit improved enzymatic activity and thermal stability, leading to enhanced protein expression and efficacy in treating cancer.
Implementation Method 1
hyaluronidase PH20 variants or fragments thereof, which comprise one or more amino acid residue substitutions in the region corresponding to the alpha-helix region and/or its linker region in wild-type PH20
Implementation Method 2
hyaluronidase which is an enzyme that hydrolyzes hyaluronic acid
Data Source
AI summary
The present invention is related to the field of protein engineering technology which increases the enzymatic activity and thermal stability of human hyaluronidase which is an enzyme that hydrolyzes hyaluronic acid; and more particularly to hyaluronidase PH20 variants or fragments thereof, which comprise one or more amino acid residue substitutions in the region corresponding to the alpha-helix region and/or its linker region in the amino acid sequence of wild-type PH20 of SEQ ID NO: 1 and in which one or more amino acid residues at the N-terminus and/or the C-terminus are selectively truncated additionally.Specifically, the present invention relates to PH20 variants or fragments thereof, which comprise one or more amino acid residue substitutions selected from the group consisting of T341A, T341C, T341G, S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the amino acid sequence of SEQ ID NO: 1, and additionally comprise the substitution of amino acids located in the alpha-helix 8 region and/or a linker region between alpha-helix 7 and alpha-helix 8 in the amino acid sequence of wild-type PH20, and in which portion(s) of amino acids located at the N-terminal and C-terminal regions are deleted.