Sortase F and its use in methods for conjugation

A method for producing a conjugate of two substrates, the method comprising the steps of:

• a. providing two substrates, each substrate being independently selected from the one or more of the groups consisting of:
• i. small molecules, and
• ii. proteins,
• and
• b. enzymatically conjugating the two substrates using sortase F enzyme of Propionibacterium acnes, which comprises an amino acid sequence according to SEQ ID NO. 1,
• wherein
• one substrate comprises a sortase F recognition motif, while
• the other substrate comprises at least one motif selected from
• a Glyn motif,
• an Alan motif, or
• a motif consisting of a mixture of Ala and Gly residues
• the motif totaling n residues, where n is an integer from ≥1 to ≤21,
• wherein the sortase F recognition motif is LPXTG (SEQ ID NO:
• 44) or LPXTGE (SEQ ID NO:
• 50), where X is any amino acid with the exception of proline,
• wherein further the reaction takes place in an aqueous reaction medium comprising a salt concentration of from about ≥0.05 to ≤3 M,
• wherein the pH in the aqueous reaction medium is adjusted to a range from ≥6.5 to ≤8.5,
• wherein the temperature in the aqueous reaction medium is adjusted to a range from ≥15 to ≤40° C., and wherein the aqueous reaction medium comprises ≤10 mM of Zn, Rb, Mg, Ni, and/or Mn cations, and comprises >0 mM and ≤10 mM of Ca, thereby producing a conjugated product of the two substrates.