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Mutations in maltose-binding protein that alter affinity and solubility properties.
Iris H Walker1, Pei-chung Hsieh, Paul D Riggs
1New England Biolabs, 240 County Rd, Ipswich, MA 01938-2723, USA.
Applied Microbiology and Biotechnology
|June 11, 2010
Summary
Researchers engineered maltose-binding protein (MBP) mutations to significantly improve maltodextrin binding and solubility. These enhanced MBP variants boost recombinant protein yield and purification efficiency.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Maltose-binding protein (MBP) from Escherichia coli is a well-established protein for engineering.
- MBP serves as both an affinity and solubility tag in recombinant protein expression.
- Previous studies demonstrated MBP's suitability for altering binding and increasing affinity.
Purpose of the Study:
- To isolate and characterize mutations in MBP that enhance binding to maltodextrins.
- To evaluate the utility of these mutant MBPs in improving the yield and solubility of fusion proteins.
- To engineer an MBP with a tighter dissociation constant for improved purification.
Main Methods:
- Random mutagenesis of MBP was employed.
- Screening for enhanced yield using a microplate-based affinity purification method.
- Measurement of dissociation constants for mutant MBPs and fusion proteins.
Main Results:
- Mutations were isolated that enhance maltodextrin binding 1.3 to 15-fold.
- Mutant MBPs improved the yield of a poorly binding fusion protein and enhanced solubility of aggregation-prone proteins.
- A combined mutation resulted in an MBP with a 10-fold tighter dissociation constant than wild-type.
Conclusions:
- Protein engineering of MBP can significantly enhance its binding affinity and solubility-enhancing properties.
- The identified mutations offer new strategies for modifying MBP function in protein expression and purification.
- These engineered MBPs hold promise for improving the production of challenging recombinant proteins.
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