Proximity-based sortase-mediated protein purification and ligation

A conjugate protein composition comprising a first protein, a second protein, and a third protein, wherein:

• (a) the first protein comprises an antibody of interest or an antigen-binding fragment thereof with
• (i) a first antibody sortase recognition construct C-terminal and connected via a linker to at least one heavy chain of the antibody or antigen-binding fragment thereof and comprising a first antibody sortase recognition sequence and a first antibody binding pair partner;
• and
• (ii) a second antibody sortase recognition construct C-terminal and connected via a linker to at least one light chain of said antibody or antigen-binding fragment thereof and comprising a second antibody sortase recognition sequence and a second antibody binding pair partner;
• (b) the second protein comprises a first binding pair partner in series with a first sortase and an affinity tag having a selective affinity for a first affinity tag resin, wherein the first antibody binding pair partner and the first binding pair partner form a first heterodimer selected from the group consisting of SpyCatcher and SpyTag;
• two complementary halves of a split intein;
• c-Jun and c-Fos;
• leucine zippers;
• split adhesin domains;
• SnoopCatcher and SnoopTag;
• S-protein and S-Tag;
• Streptavidin/Streptactin or variants thereof and Strep-tag or Strep-tag II;
• calmodulin and calmodulin binding peptide;
• and a binding pair of a dock-and-lock system, and wherein:
• said first binding pair partner is N-terminal and connected via a linker to said first sortase and said first sortase is N-terminal and connected via a linker to said first affinity tag;
• or
• said first sortase is N-terminal and connected via a linker to said first binding pair partner and said first binding pair partner is N-terminal and connected via a linker to said first affinity tag;
• or
• said first affinity tag is N-terminal and connected via a linker to said first binding pair partner and said first binding pair partner is N-terminal and connected via a linker to said first sortase;
• or
• said first affinity tag is N-terminal and connected via a linker to said first sortase and said first sortase is N-terminal and connected via a linker to said first binding pair partner;
• and
• (c) the third protein comprising a second binding pair partner in series with a second sortase and a second affinity tag, wherein said second antibody binding pair partner and said second binding pair partner comprise two protein moieties that form a second heterodimer selected from the group consisting of SpyCatcher and SpyTag;
• two complementary halves of a split intein;
• c-Jun and c-Fos;
• leucine zippers;
• split adhesin domains;
• SnoopCatcher and SnoopTag;
• S-protein and S-Tag;
• Streptavidin/Streptactin or variants thereof and Strep-tag or Strep-tag II;
• calmodulin and calmodulin binding peptide;
• and a binding pair of a dock-and-lock system, and wherein:
• said second binding pair partner is N-terminal and connected via a linker to said second sortase and said second sortase is N-terminal and connected via a linker to said second affinity tag;
• or
• said second sortase is N-terminal and connected via a linker to said second binding pair partner and said second binding pair partner is N-terminal and connected via a linker to said second affinity tag;
• or
• said second affinity tag is N-terminal and connected via a linker to said second binding pair partner and said second binding pair partner is N-terminal and connected via a linker to said second sortase;
• or
• said second affinity tag is N-terminal and connected via a linker to said second sortase and said second sortase is N-terminal and connected via a linker to said second binding pair partner.