In this section
@article{Makrydaki:2022:10.1101/2022.04.08.487553,
author = {Makrydaki, E and Donini, R and Krueger, A and Royle, K and Moya-Ramirez, I and Kuntz, DA and Rose, DR and Haslam, SM and Polizzi, K and Kontoravdi, C},
doi = {10.1101/2022.04.08.487553},
title = {Immobilised enzyme cascade for targeted glycosylation},
url = {http://dx.doi.org/10.1101/2022.04.08.487553},
year = {2022}
}
TY - JOUR
AB - <jats:title>Abstract</jats:title><jats:p>Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for <jats:underline>s</jats:underline>eq<jats:underline>u</jats:underline>ential <jats:underline>g</jats:underline>lycosyl<jats:underline>a</jats:underline>tion <jats:underline>r</jats:underline>eactions for <jats:underline>ta</jats:underline>ilo<jats:underline>r</jats:underline>ed su<jats:underline>g</jats:underline>ar s<jats:underline>t</jats:underline>ructures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation <jats:italic>in vitro</jats:italic>. This novel proof-of-concept system is enabled by immobilised enzymes produced with a “one-step immobilisation/purification” method to express, biotinylate <jats:italic>in vivo</jats:italic> and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered <jats:italic>Pichia pastoris</jats:italic>, yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised β-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures fo
AU - Makrydaki,E
AU - Donini,R
AU - Krueger,A
AU - Royle,K
AU - Moya-Ramirez,I
AU - Kuntz,DA
AU - Rose,DR
AU - Haslam,SM
AU - Polizzi,K
AU - Kontoravdi,C
DO - 10.1101/2022.04.08.487553
PY - 2022///
TI - Immobilised enzyme cascade for targeted glycosylation
UR - http://dx.doi.org/10.1101/2022.04.08.487553
ER -
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