Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule
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Frontiers in Bioengineering and Biotechnology
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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.
Como referenciar
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. Frontiers in Bioengineering and Biotechnology, v. 11, p. 1-11, 2023. DOI: 10.3389/fbioe.2023.1227212. Disponível em: http://repositorio.ipen.br/handle/123456789/34235. Acesso em: 20 Mar 2026.
Esta referência é gerada automaticamente de acordo com as normas do estilo IPEN/SP (ABNT NBR 6023) e recomenda-se uma verificação final e ajustes caso necessário.