Targeted Protein Mutagenesis for Stability Optimization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for protein stabilization through mutagenesis are limited by the large number of potential mutants generated, making it difficult to identify and evaluate those with desired stability and function, particularly due to the unpredictable effects of amino acid substitutions on protein structure and function.
Innovation Solution
A method is developed to systematically introduce specific amino acids into defined positions of proteins to generate targeted mutants, focusing on regions prone to instability such as oxidation, cyclization, and isomerization, using oligonucleotide synthesis to create libraries of mutants that are more likely to exhibit improved stability and retain functional activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If randomized mutagenesis is used to generate protein variants, then a large number of mutants can be generated, but it becomes difficult to select and evaluate those with desired stability and function
Solution Approach 1:
The patent applies local quality by focusing mutagenesis on specific regions of the protein sequence (such as complementarity determining regions or CDRs) rather than randomly mutating the entire sequence. This targeted approach generates fewer total mutants while concentrating on functionally relevant positions, thereby reducing evaluation complexity while maintaining productivity.
Solution Approach 2:
The patent segments the protein sequence into specific regions of interest (such as individual CDRs or framework regions) and performs mutagenesis on these segments independently. This allows systematic evaluation of mutants in manageable groups, reducing the overall complexity of selecting and evaluating numerous variants.
2Adaptability or versatility
If amino acid substitutions are made at multiple positions simultaneously, then more variants can be generated, but the number of potential mutations increases exponentially making identification and evaluation difficult
Solution Approach 1:
The patent segments the mutagenesis process by addressing one or a few positions at a time rather than simultaneously mutating multiple positions. This systematic approach to multi-position mutagenesis reduces the exponential growth of variants while maintaining the ability to generate versatile protein variants with improved properties.
3Ease of manufacture
If there is no reliable way to predict the effect of amino acid substitutions, then mutagenesis can be performed without guidance, but it becomes impossible to identify mutants with desired properties in a sensible manner
Solution Approach 1:
The patent applies preliminary action by using computational methods and structural information to predict the effects of potential amino acid substitutions before actually performing the mutagenesis. This allows prioritization of mutations likely to improve stability or function, making it easier to identify useful mutants while maintaining ease of performing the mutagenesis process.
Data Source
Figure 1~2
Figure 3~4
Figure 5~6
AI summary
Methods of modifying and in particular stabilizing proteins and polypeptides by which a predetermined amino acid is introduced into selected positions of said protein or polypeptide to produce a small group of mutants. The methods are based on the premise that certain amino acids play a crucial role in the stability of proteins or polypeptides. Generated mutants can then be further analysed for stability and/or function, e.g. affinity. Furthermore, appropriate mutants may be combined to result in further optimized proteins or polypeptides. In addition, stabilized example polypeptides and suitable methods to identify and/or analyse de-stabilized or stabilized proteins or polypeptides are provided. The methods can be used to study the role of specific amino acids in protein structure and function and to develop new or improved, e.g. stabilized proteins and polypeptides such as antibodies and single variable domains.