Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria， characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found， which recognize characteristic recognition sequences present in substrate proteins. Up to date， sortase A from ， a housekeeping class A sortase， is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from ， a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the genome， it is the housekeeping sortase of this organism. Sortase F from shows a behavior similar to sortases from class A in terms of pH dependence， recognition sequence and catalytic activity; furthermore， its activity is independent of bivalent ions， which contrasts to sortase A from We demonstrate that sortase F is useful for protein engineering applications， by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus， the detailed characterization presented here will not only enable the development of anti-virulence agents targeting but also provides a powerful alternative to sortase A for protein engineering applications.