We observed remarkable tolerance to loop length and sequence diversity as the resulting 38 amino acid EETI-II mutant contained 21 non-native amino acid residues distributed across two loops.
Finally, the energetics for the conversion of non-native keto acids such as 2-keto-butanoic acid, 2-keto-valeric acid and 2-keto-isovaleric acid to their corresponding alcohols were evaluated, and the thermodynamic landscapes were compared to that of the native substrate, pyruvic acid.
Importantly, we demonstrated that multiple loops of a knottin peptide, comprising 21 non-native amino acid residues, can be engineered to bind with high affinity to integrin receptors, and the resultant peptide can be used as a probe for non-invasive molecular imaging applications.
First, the laboratory mutation sets are expected to contain an overabundance of changes to alanine because of alanine scanning, which is a cost-efficient alternative to testing every possible non-native amino acid (Bromberg and Rost, 2008).
However, recombinant AMPs expressed in E. coli often include 1-2 non-native acido acid residuals at the N terminus of the target protein due to a specific linker sequence recognized by endoproteases or chemical agents, typically located between the native protein sequence and the tag [ 18].
This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids.
The resulting knottin peptide contained 21 >500%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold.
This mutability landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids.
The final KtzI protein construct has 21 non-native amino acids (including the hexahistidine tag) at its N-terminus, followed by the wild-type sequence beginning with a valine residue at position 3 (Val3).
Although historically considerable effort has been expended towards this goal (e.g. [ 6 10], reviewed in [ 11,12 ]), efficient modification of seed oil profiles to include these non-native fatty acids has until recently met with limited success.
Indeed the introduction of non-native amino acids at the N- terminus is not ideal and it is a common problem that generally exists both for the LIC and Gateway technologies.
