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

Biomol Eng. 2006 Sep;23(4):185-94. doi: 10.1016/j.bioeng.2006.02.002. Epub 2006 May 2.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics*
  • Escherichia coli / genetics
  • Gene Duplication*
  • Protein Engineering*
  • Proteins / chemistry
  • Proteins / genetics*

Substances

  • Proteins