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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold
Nimish Gera1, Mahmud Hussain, Robert C Wright
1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.
Journal of Molecular Biology
|April 26, 2011
Summary
Highly stable binding proteins were engineered from the hyperthermophilic archaeon Sulfolobus solfataricus Sso7d protein. These Sso7d mutants exhibit remarkable thermal, chemical, and pH stability, offering broad applications in biotechnology and medicine.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- The Sso7d protein from Sulfolobus solfataricus is a small, highly stable DNA-binding protein.
- Its inherent stability and lack of cysteine residues make it an attractive scaffold for protein engineering.
Purpose of the Study:
- To engineer novel, highly stable binding proteins by mutagenizing the Sso7d protein.
- To assess the binding capabilities and stability of these engineered proteins against a diverse range of targets.
Main Methods:
- Random mutagenesis of 10 amino acid residues on the DNA-binding surface of Sso7d.
- Yeast surface display technology was employed to generate and screen a library of 10^8 mutants.
- Binding affinities were determined for various targets including small molecules, peptides, and proteins.
Main Results:
- Isolation of Sso7d-derived binding proteins with nanomolar to micromolar dissociation constants for diverse targets.
- Demonstrated ability of these proteins to discriminate between closely related immunoglobulins.
- Engineered Sso7d mutants retained high thermal stability (Tm >89 °C), chemical stability, and pH stability despite extensive mutagenesis.
Conclusions:
- Mutagenesis of Sso7d is an effective strategy for generating highly stable and specific binding proteins.
- These engineered proteins exhibit excellent stability profiles, making them suitable for various biotechnological and medical applications.
- High yields of recombinant Sso7d mutants can be achieved in Escherichia coli cytoplasm.
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