Modified Chondroitin Synthase Polypeptide for High Expression and Crystallization
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Solution Overview
Problem
Current technologies do not provide a chondroitin synthase polypeptide with high expression levels, enhanced enzymatic activity, and ease of crystallization, as disclosed in existing patent documents.
Innovation Solution
A modified chondroitin synthase polypeptide with a deletion of 57 amino acid residues from the N-terminal, specifically designed to increase expression efficiency and enzymatic activity, and facilitate crystallization, along with a nucleic acid encoding this polypeptide and methods for its production and crystallization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type K4CP is used, then the polypeptide can be expressed, but the expression level is low and enzymatic activity is insufficient
Solution Approach 1:
The polypeptide is divided into functional segments: the N-terminal 57 amino acids are removed (which appear to be a signal peptide or regulatory region), and the remaining C-terminal portion (amino acids 58-686) is retained as the active enzyme. This segmentation eliminates the inhibitory or low-expression region while preserving the catalytic domain, thereby achieving high expression levels and enzymatic activity.
Solution Approach 2:
The problematic N-terminal region (57 amino acids) is extracted and removed from the wild-type K4CP sequence. This extraction eliminates the portion that hinders high-level expression and enzymatic activity, allowing the remaining polypeptide to function optimally as chondroitin synthase.
2Ease of manufacture
If wild-type K4CP is used, then the polypeptide sequence is complete, but crystallization is difficult
Solution Approach 1:
The polypeptide structure is segmented by removing the N-terminal 57 amino acids. This creates a truncated version with improved structural properties for crystallization. The remaining C-terminal domain (58-686) appears to have enhanced structural stability and regularity, facilitating crystal lattice formation while maintaining enzymatic function.
Solution Approach 2:
The amino acid sequence parameters are changed by deleting the N-terminal 57 residues. This parameter change (truncation) fundamentally alters the physical-chemical properties of the polypeptide, improving its solubility, structural homogeneity, and ability to form ordered crystal structures, thereby enabling easy crystallization for structural analysis.
3Productivity
If the polypeptide structure is modified to enhance expression and activity, then productivity increases, but the structure becomes more complex
Solution Approach 1:
Rather than adding complex modifications to enhance activity, the invention uses segmentation by removing the N-terminal 57 amino acids. This simplification paradoxically enhances enzymatic activity and expression levels, demonstrating that eliminating non-essential or inhibitory regions can improve productivity without increasing structural complexity.
Data Source
AI summary
Disclosed are: (A) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, or (B) a polypeptide comprising an amino acid sequence of SEQ ID NO:2 including deletion, substitution or addition of one or several amino acid residues and having chondroitin synthase activity; a nucleic acid encoding the polypeptide; a method for producing the polypeptide, comprising at least the steps of: (1) expressing the nucleic acid to produce the polypeptide; and (2) collecting the polypeptide produced in the step (1); and a crystal of the polypeptide. The crystal may be a monoclinic or tetragonal crystal.