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.

Sortase-mediated site-specific modification of interleukin-2 for the generation of a tumor targeting acetazolamide-cytokine conjugate

“…However, main limitations in this technology may hamper its application for commercial scale manufacturing, including the need for GMP grade SrtA and reaction substrates, the relatively high amount of SrtA required to achieve complete conversion, and the final purification step necessary to separate the conjugated product from the SrtA enzyme. The use of engineered SrtA variants 31 with increased catalytic activity, combined with the immobilization of the SrtA to solid supports may contribute to overcome some of these limitations 28 .…”

Enzyme‐mediated assembly of chimeric membrane proteins at phospholipid bilayers

“…A number of native, enzymatic‐based ligation strategies based on microbial transglutaminase from Streptomyces mobaraensis , phophopantetheine transpherase, transpeptidase sortase A (SrtA) from Staphylococcus aureus , and enzymes involved in prenylation have recently been developed, which meet this requirement. Among these approaches, SrtA enzyme has been developed into an advanced molecular biology tool that has been used in a number of applications . SrtA specifically ligates a C‐terminal LPXTG sequence on one polypeptide chain (where X is any amino acid) to a Gly‐Gly (GG) motif on the N‐terminus of another polypeptide chain .…”

“…Among these approaches, SrtA enzyme has been developed into an advanced molecular biology tool that has been used in a number of applications. [21][22][23][24] SrtA specifically ligates a C-terminal LPXTG sequence on one polypeptide chain (where X is any amino acid) to a Gly-Gly (GG) motif on the N-terminus of another polypeptide chain. 25 An SrtA variant, 26 with 140 times the activity of wild type enzyme, has also been engineered through mutation of seven residues in the native enzyme (the so-called "7M SrtA").…”

Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling

“…Wild type SrtA operates with rather slow kinetics, necessitating prolonged incubation times to obtain acceptable product yields. Several groups have contributed to optimizing the SrtA enzyme to generate variants with increased K m and K cat [ 9 , 10 ]. Of these, a pentamutant (“5M”) variant (P94R/D160N/D165A/K190E/K196T) showed a 140-fold increase in activity over wild-type SrtA, with much improved reaction rates even at low temperature [ 9 ].…”

“…Directed evolution efforts derived from wild-type and 5M SrtA yielded further improved variants with increased conjugation efficiency [ 10 ]. A D124G mutation showed a 1.6-fold increase in K cat and a 1.8-fold decrease in K m , resulting in a 3-fold improvement in catalytic efficiency over 5M SrtA.…”