Single-Domain Antibody Fusion Proteins with Alpha-Synuclein Acid Tail
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Solution Overview
Problem
Single-domain antibodies (sdAbs) expressed in the cytoplasm lack thermal stability and fail to refold after thermal denaturation, limiting their utility due to reduced melting temperature and aggregation issues.
Innovation Solution
Incorporating the acid tail of α-synuclein (ATS) into sdAbs enhances their solubility and refolding ability, even after thermal denaturation, by promoting disulfide formation and maintaining binding activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If sdAbs are expressed in the cytoplasm, then protein production is simplified, but thermal stability is lost and refolding ability is impaired
Solution Approach 1:
The patent introduces the acid tail of α-synuclein (ATS) as an intermediary element that mediates between the cytoplasmic expression environment and the need for thermal stability. The ATS sequence acts as a chaperone-like factor that enables proper folding and refolding of sdAbs expressed in the cytoplasm, resolving the contradiction between ease of manufacture and thermal stability.
Solution Approach 2:
The patent creates a composite protein structure by fusing the sdAb with the ATS sequence. This composite fusion protein combines the binding functionality of the sdAb with the stabilizing properties of the ATS tail, achieving both ease of cytoplasmic expression and thermal stability in a single molecular construct.
2Quantity of substance
If sdAbs are heated above melting temperature, then solubility may improve, but functional activity is lost due to aggregation
Solution Approach 1:
The ATS sequence provides beforehand cushioning by acting as a protective element that prevents aggregation before it occurs. The acidic residues in the ATS tail create electrostatic repulsion that cushions against the aggregation of hydrophobic regions during thermal stress, allowing the protein to maintain solubility and functionality even when heated above its melting temperature.
3Reliability
If periplasmic location tag is used, then disulfide bond formation is improved, but device complexity increases
Solution Approach 1:
The patent extracts and removes the periplasmic location tag from the protein construct, achieving disulfide bond formation without requiring this additional element. The ATS sequence itself provides the necessary stabilizing function, allowing the sdAb to form disulfide bonds in the cytoplasmic environment without the complexity of periplasmic targeting signals.
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 ATS fusion proteins demonstrate improved solubility and refolding capabilities, maintaining functional activity even when heated above their melting temperature, thus overcoming the limitations of cytoplasmic expression and aggregation.
Implementation Method 1
Incorporating the acid tail of α-synuclein (ATS) into sdAbs enhances their solubility and refolding ability, even after thermal denaturation, by promoting disulfide formation and maintaining binding activity.
Implementation Method 2
Single-domain antibodies (sdAbs) expressed in the cytoplasm lack thermal stability and fail to refold after thermal denaturation
Implementation Method 3
the fusion protein is free of a periplasmic location tag... demonstrate improved solubility and refolding capabilities, maintaining functional activity even when heated above their melting temperature
Data Source
AI summary
A single-domain antibody (sdAb) is produced by causing a bacteria to express the sdAb into cytoplasm of the bacteria, wherein the sdAb is expressed as a fusion protein with the acid tail of α-synuclein. In embodiments, the protein is free of a periplasmic location tag. Such antibodies have the unexpected ability to refold after thermal denaturation.