Mutant TSHR Thermostability via Rational Scanning Mutagenesis
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Solution Overview
Problem
Thyroid stimulating hormone receptors (TSHR) and their fragments, such as TSHR260, have poor stability during purification, which hinders the production of highly purified forms and their use in assays for detecting autoantibodies associated with autoimmune thyroid diseases.
Innovation Solution
Introducing specific single and combined point mutations into the TSHR and TSHR260 using rational-scanning mutagenesis to enhance thermostability, allowing for improved protein stability and retention of biological activity, enabling more effective detection and purification of TSHR autoantibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type TSHR is used for purification and assay preparation, then the native structure is preserved, but the protein exhibits poor stability during purification
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (mutations) into the TSHR protein sequence. These mutations modify the protein's physical and chemical properties to enhance thermostability and structural integrity during purification processes, while maintaining the receptor's ability to bind autoantibodies for diagnostic assays
2Stability of the object's composition
If mutations are introduced to improve thermostability, then protein stability increases, but there is a risk of losing biological activity
Solution Approach 1:
The patent applies local quality by introducing mutations at specific, strategically selected positions within the TSHR protein structure. These localized changes are designed to stabilize particular structural regions (such as the extracellular domain) without disrupting the overall folding or the critical autoantibody-binding sites, thus maintaining biological activity while improving thermostability
3Stability of the object's composition
If multiple point mutations are combined to further improve stability, then thermostability is enhanced, but the complexity of the mutation combination increases
Solution Approach 1:
The patent applies merging by combining multiple beneficial point mutations into a single integrated TSHR construct. This allows the cumulative stabilizing effects of individual mutations to work synergistically, achieving significantly enhanced thermostability and purification stability while maintaining a manageable level of complexity through rational design
Data Source
AI summary
A mutant thyroid stimulating hormone receptor (TSHR) or fragment thereof comprises one or more mutations, wherein the mutant TSHR has increased thermostability with respect to the equivalent wild type TSHR or fragment. The one or more mutation is preferably within the extracellular leucine-rich repeat domain (LRD) of the TSHR, or within residues 22 to 260 (TSHR260) of the TSHR, or may be in the transmembrane domain (TMD), A mutant TSHR or fragment thereof of the invention preferably consists of, or consists essentially of, the subdomain TSHR260 of the TSHR receptor. A mutant TSHR or fragment thereof according to the invention has a greater thermostability than the equivalent wild type TSHR or fragment as determined by its half-life at a given temperature, and can be purified whilst retaining activity. A mutant TSHR or fragment thereof according to the invention may also be deglycosylated whilst retaining activity. Methods, kits and uses employing the mutant TSHR or fragment thereof according to the invention are also provided.