Design of novel FN3 domains with high stability by a consensus sequence approach

Abstract

The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robustly expressed in Escherichia coli, soluble and highly stable, with melting temperatures of 89 and 78°C, respectively. X-ray crystallography was used to confirm that the consensus approach led to a structure consistent with the FN3 design despite having only low-sequence identity to natural FN3 domains. The ability of the Tenascin consensus domain to withstand mutations in the loop regions connecting the β-strands was investigated using alanine scanning mutagenesis demonstrating the potential for randomization in these regions. Finally, rational design was used to produce point mutations that significantly increase the stability of one of the consensus domains. Together our data suggest that consensus FN3 domains have potential utility as alternative scaffold therapeutics.

Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

“…However, unlike DARPins we are introducing 18 variable amino acid residues by replacing two short loops of a non-repeat protein scaffold and so the retention of such high thermostability is notable. Fibronectins and the leucine-rich repeat-based Repebodies have reported melting temperatures of around 90°C (Jacobs et al , 2012) and 85°C (Lee et al , 2012), respectively.…”

“…While any natural protein is expected only to have evolved the level of stability required for it to efficiently perform its function, a consensus protein reinforces structural stability. This approach has been successfully used to improve the thermostability of enzymes (Lehmann et al , 2000; Komor et al , 2012), antibodies (Knappik et al , 2000) as well as artificial binding proteins (Main et al , 2003; Forrer et al , 2004; Jacobs et al , 2012). …”

Creating a Homeodomain with High Stability and DNA Binding Affinity by Sequence Averaging

“…14,18 Most recently, the O’Neil and Buckle groups have designed consensus FN3 domains and a consensus serpin that adopt their respective folds and retain high thermostability. 19–21 …”

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