Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates
S.1-10 Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuE...
Published in: | Journal of Biotechnology |
---|---|
Main Authors: | , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
2022
|
Subjects: | |
Online Access: | https://publica.fraunhofer.de/handle/publica/415279 https://doi.org/10.1016/j.jbiotec.2022.01.001 |
id |
ftfrauneprints:oai:publica.fraunhofer.de:publica/415279 |
---|---|
record_format |
openpolar |
spelling |
ftfrauneprints:oai:publica.fraunhofer.de:publica/415279 2023-07-16T03:54:52+02:00 Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates Alaneed, Razan Naumann, Marcel Pietzsch, Markus Kressler, Jörg 2022 https://publica.fraunhofer.de/handle/publica/415279 https://doi.org/10.1016/j.jbiotec.2022.01.001 en eng 10.1016/j.jbiotec.2022.01.001 Journal of biotechnology doi:10.1016/j.jbiotec.2022.01.001 https://publica.fraunhofer.de/handle/publica/415279 Amine-grafted poly(D-sorbitol adipate) biodegradability Erythroppietin thermoresistant microbial transglutaminase rHuEPO conjugate 610 660 620 journal article 2022 ftfrauneprints https://doi.org/10.1016/j.jbiotec.2022.01.001 2023-06-25T23:43:23Z S.1-10 Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a Tm of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans. 346 Article in Journal/Newspaper antartic* Publikationsdatenbank der Fraunhofer-Gesellschaft Journal of Biotechnology 346 1 10 |
institution |
Open Polar |
collection |
Publikationsdatenbank der Fraunhofer-Gesellschaft |
op_collection_id |
ftfrauneprints |
language |
English |
topic |
Amine-grafted poly(D-sorbitol adipate) biodegradability Erythroppietin thermoresistant microbial transglutaminase rHuEPO conjugate 610 660 620 |
spellingShingle |
Amine-grafted poly(D-sorbitol adipate) biodegradability Erythroppietin thermoresistant microbial transglutaminase rHuEPO conjugate 610 660 620 Alaneed, Razan Naumann, Marcel Pietzsch, Markus Kressler, Jörg Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
topic_facet |
Amine-grafted poly(D-sorbitol adipate) biodegradability Erythroppietin thermoresistant microbial transglutaminase rHuEPO conjugate 610 660 620 |
description |
S.1-10 Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a Tm of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans. 346 |
format |
Article in Journal/Newspaper |
author |
Alaneed, Razan Naumann, Marcel Pietzsch, Markus Kressler, Jörg |
author_facet |
Alaneed, Razan Naumann, Marcel Pietzsch, Markus Kressler, Jörg |
author_sort |
Alaneed, Razan |
title |
Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
title_short |
Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
title_full |
Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
title_fullStr |
Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
title_full_unstemmed |
Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
title_sort |
microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates |
publishDate |
2022 |
url |
https://publica.fraunhofer.de/handle/publica/415279 https://doi.org/10.1016/j.jbiotec.2022.01.001 |
genre |
antartic* |
genre_facet |
antartic* |
op_relation |
10.1016/j.jbiotec.2022.01.001 Journal of biotechnology doi:10.1016/j.jbiotec.2022.01.001 https://publica.fraunhofer.de/handle/publica/415279 |
op_doi |
https://doi.org/10.1016/j.jbiotec.2022.01.001 |
container_title |
Journal of Biotechnology |
container_volume |
346 |
container_start_page |
1 |
op_container_end_page |
10 |
_version_ |
1771541290436001792 |