Specific and High Affinity Binding Proteins Comprising Modified SH3 Domains of FYN Kinase

A library comprising a plurality of recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:

• 1, one or more of said derivatives having a specific binding affinity to a protein or peptide that is not a natural SH3 binding ligand,
• wherein substantially each of the derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 has an amino acid sequence complying with the following requirements:
• (i) it has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:
• 1, and
• (ii) it has at least 90% identity to the amino acid of SEQ ID NO:
• 1 outside the src and RT loops;
• (iii, a) at least one amino acid in or positioned up to two amino acids adjacent to the RT loop of SEQ ID NO:
• 1 is substituted, deleted or added,
• (iii, b) at least one amino acid in or positioned up to two amino acids adjacent to the src loop of SEQ ID NO:
• 1 is substituted, deleted or added;
• or
• (iii, c) at least one amino acid in or positioned up to two amino acids adjacent to the RT loop of SEQ ID NO:
• 1 is substituted, deleted or added, and at least one amino acid in or positioned up to two amino acids adjacent to the src loop of SEQ ID NO:
• 1 is substituted, deleted or added;
• wherein the src loop is located at amino acid positions 31 to 34 of SEQ ID NO:
• 1, and the RT loop is located at amino acid positions 12 to 17 of SEQ ID NO:
• 1.

A method for selecting from a library comprising recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:

• 1 one or more of said derivatives having a specific binding affinity to a protein or peptide, said method comprising the steps of
• (a) contacting the library comprising derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 with the protein or peptide
• under conditions and for a time sufficient to permit the derivatives and the protein or peptide to interact;
• and
• (b) selecting from the library one or more derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 having a specific binding affinity to the protein or peptide,
• wherein the protein or peptide is not a natural SH3 binding ligand, and
• wherein each of the derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 having a specific binding affinity to the protein or peptide has an amino acid sequence complying with the following requirements:
• (i) it has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:
• 1, and
• (ii) it has at least 90% identity to the amino acid of SEQ ID NO:
• 1 outside the src and RT loops;
• (iii, a) at least one amino acid in or positioned up to two amino acids adjacent to the RT loop of SEQ ID NO:
• 1 is substituted, deleted or added, and
• (iii, b) at least one amino acid in or positioned up to two amino acids adjacent to the src loop of SEQ ID NO:
• 1 is substituted, deleted or added;
• wherein the src loop is located at amino acid positions 31 to 34 of SEQ ID NO:
• 1, and the RT loop is located at amino acid positions 12 to 17 of SEQ ID NO:
• 1.

A method for the production of a library comprising a plurality of recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:

• 1, one or more of said derivatives having a specific binding affinity to a protein or peptide that is not a natural SH3 binding ligand, said method comprising the steps of
• (a, i) generating recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 by substitution, addition, and/or deletion of at least one amino acid in or positioned up to two amino acids adjacent to the RT loop of SEQ ID NO:
• 1;
• (a, ii) generating recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 by substitution, addition, and/or deletion of at least one amino acid in or positioned up to two amino acids adjacent to the src loop of SEQ ID NO:
• 1;
• or
• (a, iii) generating recombinant derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 by substitution, addition, and/or deletion of at least one amino acid in or positioned up to two amino acids adjacent to the RT loop and by substitution, addition, and/or deletion of at least one amino acid in or positioned up to two amino acids adjacent to the src loop of SEQ ID NO:
• 1;
• (b) constructing a library comprising the recombinant derivatives of the SH3 domain generated in step (a),
• wherein the derivatives of the SH3 domain of the Fyn kinase of SEQ ID NO:
• 1 retain at least 80% sequence identity to the amino acid sequence of SEQ ID NO:
• 1, and retain at least 90% sequence identity to the amino acid of SEQ ID NO:
• 1 outside the src and RT loops;
• and
• wherein the src loop is located at amino acid positions 31 to 34 of SEQ ID NO:
• 1, and the RT loop is located at amino acid positions 12 to 17 of SEQ ID NO:
• 1.