Cargando…

Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolys...

Descripción completa

Detalles Bibliográficos
Autores principales: Lima, G. C., Chura-Chambi, R. M., Morganti, L., Silva, V. J., Cabral-Piccin, M. P., Rocha, V., Medina, T. S., Ramos, R. N., Luz, D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10426796/
https://www.ncbi.nlm.nih.gov/pubmed/37588136
http://dx.doi.org/10.3389/fbioe.2023.1227212
_version_ 1785090133495644160
author Lima, G. C.
Chura-Chambi, R. M.
Morganti, L.
Silva, V. J.
Cabral-Piccin, M. P.
Rocha, V.
Medina, T. S.
Ramos, R. N.
Luz, D.
author_facet Lima, G. C.
Chura-Chambi, R. M.
Morganti, L.
Silva, V. J.
Cabral-Piccin, M. P.
Rocha, V.
Medina, T. S.
Ramos, R. N.
Luz, D.
author_sort Lima, G. C.
collection PubMed
description Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.
format Online
Article
Text
id pubmed-10426796
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-104267962023-08-16 Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule Lima, G. C. Chura-Chambi, R. M. Morganti, L. Silva, V. J. Cabral-Piccin, M. P. Rocha, V. Medina, T. S. Ramos, R. N. Luz, D. Front Bioeng Biotechnol Bioengineering and Biotechnology Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints. Frontiers Media S.A. 2023-07-31 /pmc/articles/PMC10426796/ /pubmed/37588136 http://dx.doi.org/10.3389/fbioe.2023.1227212 Text en Copyright © 2023 Lima, Chura-Chambi, Morganti, Silva, Cabral-Piccin, Rocha, Medina, Ramos and Luz. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Lima, G. C.
Chura-Chambi, R. M.
Morganti, L.
Silva, V. J.
Cabral-Piccin, M. P.
Rocha, V.
Medina, T. S.
Ramos, R. N.
Luz, D.
Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title_full Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title_fullStr Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title_full_unstemmed Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title_short Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
title_sort recombinant human tim-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10426796/
https://www.ncbi.nlm.nih.gov/pubmed/37588136
http://dx.doi.org/10.3389/fbioe.2023.1227212
work_keys_str_mv AT limagc recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT churachambirm recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT morgantil recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT silvavj recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT cabralpiccinmp recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT rochav recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT medinats recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT ramosrn recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule
AT luzd recombinanthumantim3ectodomainexpressedinbacteriaandrecoveredfrominclusionbodiesasastableandactivemolecule