Mutations in maltose-binding protein that alter affinity and solubility properties
Open Access
- 10 June 2010
- journal article
- applied genetics-and-molecular-biotechnology
- Published by Springer Science and Business Media LLC in Applied Microbiology and Biotechnology
- Vol. 88 (1), 187-197
- https://doi.org/10.1007/s00253-010-2696-y
Abstract
Maltose-binding protein (MBP) from Escherichia coli has been shown to be a good substrate for protein engineering leading to altered binding (Marvin and Hellinga, Proc Natl Acad Sci U S A 98:4955–4960, 2001a) and increased affinity (Marvin and Hellinga, Nat Struct Biol 8:795–798, 2001b; Telmer and Shilton, J Biol Chem 278:34555–34567, 2003). It is also used in recombinant protein expression as both an affinity tag and a solubility tag. We isolated mutations in MBP that enhance binding to maltodextrins 1.3 to 15-fold, using random mutagenesis followed by screening for enhanced yield in a microplate-based affinity purification. We tested the mutations for their ability to enhance the yield of a fusion protein that binds poorly to immobilized amylose and their ability to enhance the solubility of one or more aggregation-prone recombinant proteins. We also measured dissociation constants of the mutant MBPs that retain the solubility-enhancing properties of MBP and combined two of the mutations to produce an MBP with a dissociation constant 10-fold tighter than wild-type MBP. Some of the mutations we obtained can be rationalized based on the previous work, while others indicate new ways in which the function of MBP can be modified.Keywords
This publication has 19 references indexed in Scilit:
- Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteinsBiochemical and Biophysical Research Communications, 2007
- Crystal structure of a catalytic intermediate of the maltose transporterNature, 2007
- Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMRNature, 2007
- Insights into the Conformational Equilibria of Maltose-binding Protein by Analysis of High Affinity MutantsPublished by Elsevier BV ,2003
- Structural Evidence for a Dominant Role of Nonpolar Interactions in the Binding of a Transport/Chemosensory Receptor to Its Highly Polar Ligands,Biochemistry, 2001
- Single amino acid substitutions on the surface ofEscherichia colimaltose‐binding protein can have a profound impact on the solubility of fusion proteinsProtein Science, 2001
- Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fusedProtein Science, 1999
- Direct Random Mutagenesis of Gene-Sized DNA Fragments Using Polymerase Chain ReactionAnalytical Biochemistry, 1995
- A Procedure for Renaturation and Purification of the Extracellular Serratia marcescens Nuclease from Genetically Engineered Escherichia coliProtein Expression and Purification, 1994
- Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxisBiochemistry, 1992