Engineered Alpha Klotho Polypeptides for Yield and Stability
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Solution Overview
Problem
Existing technologies face challenges in producing alpha klotho polypeptides that exhibit effective bioactivity and can be produced efficiently at scale, particularly due to issues with yield and stability.
Innovation Solution
Engineered alpha klotho polypeptides are developed, lacking a transmembrane domain and featuring specific amino acid substitutions, such as C521S in the KL2 domain and C-terminal tail modifications, optionally with a stabilization moiety linked via a protease-cleavable linker, to enhance activity, yield, and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type human KL is used, then the protein has natural bioactivity, but production yield and stability are insufficient
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (C521S in KL2 domain, C970S or deletion in C-terminal tail) to modify the protein's biochemical properties. These changes optimize folding, reduce aggregation, and improve both production yield and stability while preserving bioactivity through the conserved FGF23 coreceptor binding interface.
Solution Approach 2:
The patent employs composite materials by fusing the alpha klotho moiety with stabilization moieties (such as Fc, HSA, or other protein stabilizers) via linkers. This creates a chimeric protein where the klotho functional domain maintains its biological activity while the stabilization domain enhances structural stability and production yield.
2Reliability
If the protein is engineered for enhanced activity, then bioactivity improves, but production complexity increases
Solution Approach 1:
The patent applies segmentation by dividing the protein into distinct functional modules: the alpha klotho moiety (amino acids 34-950) containing the FGF23 coreceptor binding interface, and optional stabilization moieties (amino acids 951-...). This modular design allows independent optimization of each component and simplifies production by enabling separate expression and purification steps.
Solution Approach 2:
The patent extracts the essential functional element (the KL2 domain with FGF23 binding interface) while removing or modifying problematic regions (transmembrane domain, C-terminal cysteine at position 970). This extraction approach maintains bioactivity by preserving the critical binding interface while eliminating factors that compromise stability and yield.
3Stability of the object's composition
If C-terminal cysteine is present at position 970, then natural protein structure is maintained, but aggregation and reduced stability occur
Solution Approach 1:
The patent converts the harmful effect of the C-terminal cysteine at position 970 (which causes aggregation and reduces stability) into a beneficial modification by substituting it with serine (C970S) or deleting it entirely. This modification eliminates the aggregation-prone cysteine while preserving the functional integrity of the protein through the stabilized alternative sequence.
Data Source
AI summary
The present disclosure provides engineered alpha klotho polypeptides and methods of their production and use. Soluble alpha klotho polypeptides disclosed herein comprise an alpha klotho moiety having one or more mutations for enhancing activity, production yield, and/or stability relative to human alpha klotho. The disclosure further provides pharmaceutical compositions comprising the engineered alpha klotho polypeptides, and methods of use of the engineered alpha klotho polypeptides treatment methods, including treatment of age-related conditions and kidney disease. Also disclosed are nucleic acids encoding the engineered alpha klotho polypeptides, recombinant cells that express the engineered alpha klotho polypeptides, and methods of producing the engineered alpha klotho polypeptides.