title: Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency.
abstract: Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme's poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China.
authors.forename: Long
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, MA 02140, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Cohen
authors.affiliation: School of Life Sciences, Nanjing University, China.
authors.forename: Xiaoda
authors.initials: X
authors.lastname: Song
authors.affiliation: Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China.
authors.forename: Aishan
authors.initials: A
authors.lastname: Zhao
authors.affiliation: Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China.
authors.forename: Zi
authors.initials: Z
authors.lastname: Ye
authors.affiliation: Department of Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, MA 02140, USA.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Feulner
authors.affiliation: Department of Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, MA 02140, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Doonan
authors.affiliation: Department of Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, MA 02140, USA.
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.affiliation: Department of Global Biotherapeutics Technologies, Pfizer Inc., Cambridge, MA 02140, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Lin
authors.affiliation: Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Peking University, Beijing 100871, China.|Peking-Tsinghua Center for Life Sciences, Beijing, China.
authors.forename: Peng R
authors.initials: PR
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics; D000906:Antibodies / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics; D020125:Mutation, Missense; D011092:Polyethylene Glycols / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D011092:Polyethylene Glycols; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases