Toolkit for the production of post-translationally modified proteins

A method of phosphorylating a substrate protein of interest comprising contacting a fusion protein and a substrate protein for a period of time sufficient to allow for phosphorylation of the substrate protein by the fusion protein to occur, wherein (a) the fusion protein comprises a kinase catalytic domain, a targeting domain and a first linker, wherein the targeting domain is a SH3 (src Homology 3) domain and the kinase catalytic domain and the targeting domain are joined by the first linker;

• and (b) the substrate protein comprises a polypeptide of interest, a sequence that interacts with the targeting domain of the fusion protein, a protease cleavage site, and an optional second linker;
• wherein (i) the sequence that interacts with the targeting domain is a polyproline sequence selected from the group consisting of:
• SEQ ID NO:
• 2, SEQ ID NO:
• 3, SEQ ID NO:
• 4, a variant with at least 80% sequence identity to SEQ ID NO:
• 2, 3 or 4 that is capable of binding to an SH3 domain and a sequence of about 5 to about 20 amino acids in length and contains the sequence proline-Xaa-Xaa-proline, wherein Xaa is any amino acid;
• (ii) the protease cleavage site is between the polypeptide of interest and the second linker when the second linker is present, and between the polypeptide of interest and the sequence that interacts with the targeting domain when the second linker is not present;
• and (iii) wherein the first linker contains repeats of two, oppositely-charged, amino acids, and the first linker is about 10 to about 30 amino acids in length and/or the second linker contains repeats of two, oppositely-charged, amino acids, and the second linker is about 10 to about 30 amino acids in length.