Engineering of beta-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat.

摘要

The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence-structure and structure-function relationships. While beta-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design beta-proteins than alpha-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of beta-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the beta-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.