Proteins containing multiple, different unnatural amino acids and methods of making and using such proteins
The invention relates generally to methods of producing a protein comprising a first unnatural amino acid (UAA) and a second, different UAA, and proteins comprising a first UAA and a second, different UAA.
1 . A protein expressed in a mammalian cell, wherein the protein comprises two distinct unnatural amino acids incorporated into the protein at two site-specific locations, wherein the first unnatural amino acid (UAA) is a tryptophan analog selected from 5-HTP and 5-AzW and the second UAA is a leucine analog selected from LCA and Cys-5-N3.
2 . The protein of claim 1 , wherein a detectable label or molecule is covalently coupled to the protein via the first UAA.
3 . The protein of claim 1 , wherein a detectable label or molecule is covalently coupled to the protein via the second UAA.
4 . The protein of claim 3 , wherein the detectable label or molecule coupled to the protein via the first UAA is different from the detectable label or molecule coupled to the protein via the second UAA.
5 . The protein of claim 1 , wherein the protein is an antibody or an antigen-binding fragment thereof, a bispecific antibody, nanobody, affibody, viral protein, chemokine, cytokine, antigen, blood coagulation factor, hormone, growth factor, enzyme or a cell signaling protein.
6 . The protein of claim 5 , wherein the protein is an antibody or an antigen-binding fragment thereof.
7 . The antibody of claim 6 , wherein the antibody is a human IgG antibody, and wherein the first UAA is incorporated into a light chain of the antibody and the second UAA is incorporated into a heavy chain of the antibody.
8 . The antibody of claim 7 , wherein the first UAA is either 5-HTP or 5-AzW and the second UAA is either LCA or Cys-5-N3.
9 . The antibody of claim 8 , wherein the first UAA and the second UAA are chemically conjugated to a detectable label, a small molecule, or a drug.