SlpA Chaperone Fusion for Protein Thermal Stability
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Solution Overview
Problem
Current chaperone fusion modules, such as SlyD, have limited thermal stability, leading to thermal-induced aggregation of proteins during storage or shipment, which compromises the stability and solubility of target proteins, especially at elevated temperatures.
Innovation Solution
The use of E. coli SlpA as a fusion partner, which exhibits high intrinsic stability and retains its native fold up to 56°C, thereby preventing heat-induced aggregation and maintaining the solubility of target proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If SlyD chaperone is used as fusion partner, then solubility of target protein is improved, but thermal stability deteriorates leading to aggregation at elevated temperatures
Solution Approach 1:
The patent changes the chaperone protein parameter from SlyD to SlpA, which has different thermal stability properties. SlpA maintains its chaperone function while providing superior thermal stability up to 56°C, resolving the contradiction between solubility enhancement and thermal stability.
Solution Approach 2:
The invention creates a composite fusion protein system combining SlpA chaperone with target protein. This composite structure leverages the chaperone domain's ability to bind hydrophobic surfaces and prevent aggregation, while SlpA's inherent thermostability protects the fusion protein at elevated temperatures.
2Stability of the object's composition
If chaperone fusion modules are used, then solubility of target protein is improved, but protein aggregation increases under thermal stress
Solution Approach 1:
SlpA acts as an intermediary chaperone protein that binds to hydrophobic surfaces of the target protein. This intermediary function prevents direct aggregation of target proteins under thermal stress, mediating protective interactions that maintain solubility and prevent harmful aggregation.
Solution Approach 2:
The SlpA chaperone provides preliminary protection against thermal-induced aggregation by binding to aggregation-prone regions of the target protein before heat stress occurs. This preemptive binding prevents the formation of aggregation-prone intermediates during thermal stress.
3Temperature
If SlpA is used as fusion partner, then thermal stability is improved, but complexity of fusion protein construction increases
Solution Approach 1:
SlpA serves multiple functions in the fusion protein: it acts as a chaperone domain for proper folding, provides thermal stability up to 56°C, and maintains solubility. This multi-functionality reduces the need for additional separate components, simplifying the overall construction despite the initial complexity.
Data Source
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AI summary
The present invention relates to a recombinant DNA molecule encoding a fusion protein comprising a SIpA chaperone and a target polypeptide wherein human FK506 binding proteins (FKBPs) are excluded as target polypeptides, a corresponding expression vector encoding said fusion protein as well as host cells transformed with said expression vector. Another aspect of the invention is a method for producing said fusion protein as well as a recombinantly produced fusion protein comprising a SlpA chaperone and a target polypeptide. A further aspect of the invention is the use of the recombinantly produced fusion protein as a binding partner or as a means for the reduction of interferences in an immunoassay. Further the invention relates to the use of the recombinantly produced fusion protein for immunization of laboratory animals in order to produce antibodies and to the use of the recombinantly produced fusion protein in the production of a vaccine. Yet another aspect is a method for the detection of an analyte in an immunoassay using a recombinantly produced fusion protein as well as a reagent kit containing a recombinantly produced fusion protein comprising a SlpA chaperone and a target polypeptide. A further aspect of the invention concerns the use of SlpA for the reduction of interferences in an immunoassay and its use as an additive in protein formulations and as a folding helper in biotechnological applications.