Mutant Endonuclease Yeast Expression Uniformity
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Solution Overview
Problem
Current methods for producing wild-type endonucleases from Serratia marcescens using yeast hosts result in non-uniform chemical structures due to the addition of N-linked sugar chains during secretory production, affecting the enzyme's uniformity and activity.
Innovation Solution
A mutant polypeptide of the wild-type endonuclease is produced using a yeast host expression system without the addition of N-linked sugar chains, specifically modifying residues Asn20 and Asn33 to prevent sugar chain attachment, ensuring uniformity and maintaining endonuclease activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If secretory production of endonuclease is performed using a yeast host, then the production capability and secretion efficiency are improved, but the chemical structure uniformity deteriorates due to non-uniform N-linked sugar chain addition
Solution Approach 1:
The patent extracts and eliminates the N-linked sugar chains from the endonuclease molecule through enzymatic deglycosylation treatment. This removes the source of structural heterogeneity while preserving the core protein structure and enzymatic activity, thereby resolving the contradiction between maintaining secretion efficiency and achieving chemical structure uniformity
Solution Approach 2:
The patent changes the post-translational modification state of the protein by removing N-linked sugar chains through controlled enzymatic treatment. This parameter change (from glycosylated to deglycosylated state) transforms the heterogeneous mixture into a uniform product while maintaining the advantages of yeast secretory production
2Reliability
If N-linked sugar chains are added during secretory production in yeast, then the protein secretion and stability are improved, but the enzyme activity uniformity deteriorates
Solution Approach 1:
The patent removes N-linked sugar chains through enzymatic deglycosylation to eliminate the source of activity variation. By extracting the heterogeneous sugar moieties while retaining the stable core protein structure, the method achieves uniform enzyme activity while preserving the stability benefits of secretory production
Solution Approach 2:
The patent applies deglycosylation treatment as a preliminary purification step before final enzyme preparation. This preliminary action removes structural heterogeneity early in the process, ensuring uniform enzyme activity in the final product while maintaining the stability advantages of yeast-produced secreted 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 approach allows for the production of a uniformly structured mutant polypeptide with high endonuclease activity, overcoming the issue of non-uniformity and ensuring consistent enzymatic performance.
Implementation Method 1
the endonuclease produced comprises the N-linked sugar chain added thereto
Implementation Method 2
having endonuclease activity
Data Source
AI summary
A polypeptide includes an amino acid sequence selected from the group consisting of: an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1; and an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution, deletion, and/or addition of one or more amino acid residues. A yeast host expressing the polypeptide in a secretory production system does not add an N-linked sugar chain to the polypeptide, and the polypeptide has endonuclease activity.