Intrabodies targeting post-translational modifications of native proteins and method for obtaining them

A method for selecting an immunoglobulin able to bind in an intracellular environment to a post-translationally modified target or for determining the ability of an immunoglobulin to bind in an intracellular environment to a post-translationally modified target, wherein the post-translational modification is acetylation or phosphorylation:

• said method comprising the steps of:
• a) providing a first nucleotide sequence encoding an intracellular immunoglobulin which is associated with a first molecule;
• and
• b) providing a second nucleotide sequence encoding a second molecule and an intracellular target which:
• is associated with an enzyme that modifies the target in vivo, or
• is subjected to a direct site-specific genetic encoding of the Post-Translational Modifications (PTM) into the target protein,
• wherein the enzyme is an acetyl transferase or a tau kinase, and
• wherein said first and second molecules are separable domains of a reporter molecule;
• and
• c) expressing said first nucleotide sequence together with said second nucleotide sequence in an intracellular environment,
• wherein binding of said immunoglobulin with said target leads to stable interaction of the first molecule and second molecule, thus producing a detectable reporter molecule that generates a signal, and
• d) detecting said signal from said detectable reporter molecule, wherein said detection of a signal is indicative of stable binding activity between said immunoglobulin and said target in the intracellular environment;
• e) isolating those immunoglobulins that stably bind to the target and optionally
• f) selecting those immunoglobulins that do not bind to target that is not post-translationally modified;
• wherein the first molecule is the activation domain of VP16 and the second molecule is the DNA-binding domain of LexA
• and wherein the immunoglobulin is selected from the group consisting of:
• an intact immunoglobulin, a Fv, a scFv (single chain Fv fragment), a Fab, a F(ab′)2, an “antibody-like” domain, an “antibody-mimetic” domain, and a single antibody domain (VH domain or VL domains).