Linker Peptides Reducing Xylose Addition for Homogeneity
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Solution Overview
Problem
Existing protein engineering techniques face challenges in producing homogeneous polypeptide preparations due to post-translational modifications, particularly the addition of xylose residues, which affect the homogeneity, stability, and aggregation of proteins.
Innovation Solution
Incorporating linker peptides with specific amino acid sequences such as (GGGGA)n GGGGS, (GGGGQ)2 GGGGS, (GGGPS)2 GGGGS, and GGGGS(PGGGS)2 that lack the GSG sequence, reducing the ability of enzymes like xylosyltransferase to add xylose, thereby enhancing protein homogeneity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If standard linker peptides containing GSG sequence are used, then flexibility and unstructured conformation are achieved, but post-translational modifications (xylose addition) occur reducing homogeneity
Solution Approach 1:
The invention extracts and removes the problematic GSG sequence from the linker peptide design. By deliberately excluding this tripeptide motif, the patent eliminates the substrate recognition site for xylosyltransferase while preserving the linker's essential flexible and unstructured conformational properties through alternative glycine and serine arrangements
Solution Approach 2:
The invention applies local quality modification by specifically altering the amino acid sequence at the linker region where post-translational modifications occur. The linker maintains its overall flexible nature through glycine and serine residues but introduces local sequence variations that prevent enzyme recognition and xylose addition, thereby achieving site-specific modification prevention without compromising global linker function
2Reliability
If traditional Gly-Ser linkers are used, then domain connection and flexibility are achieved, but aggregation increases and pH stability decreases
Solution Approach 1:
The invention changes the primary sequence parameters of the linker peptide by modifying the specific arrangement and composition of glycine and serine residues. These parameter changes in the amino acid sequence lead to improved physical-chemical properties including reduced aggregation倾向 and enhanced pH stability, while maintaining the linker's flexible conformational characteristics
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 use of these linker peptides results in polypeptides with reduced aggregation, increased pH stability, and improved homogeneity by minimizing post-translational modifications, leading to more consistent protein products.
Implementation Method 1
the novel linker peptides disclosed herein reduce the ability of enzymes to link carbohydrate adducts to polypeptides comprising these linker peptides, e.g., reduce the ability of xylosyltransferase to link xylose to polypeptides
Data Source
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AI summary
The invention is based, at least in part, on the finding that linker peptides which lack the amino acid sequence GSG reduce or eliminate the addition of posttranslational modifications to the polypeptides which comprise them. More specifically, the novel linker peptides disclosed herein reduce the ability of enzymes to link carbohydrate adducts to polypeptides comprising these linker peptides, e.g., reduce the ability of xylosyltransferase to link xylose to polypeptides. These novel linker peptides, molecules comprising same, and methods of their use are described.