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.