Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3
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Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by
bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene
LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins
from the Complement System. However, the production of this recombinant protein using traditional
refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs)
solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of
the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the
aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of
these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and
alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH
of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not
reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a
yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified
thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against
complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of
thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to
establish efficient HHP-based processes for refolding of bioactive proteins from IBs.
Como referenciar
CHURA-CHAMBI, ROSA M.; FRAGA, TATIANA R.; SILVA, LUDMILA B. da; YAMAMOTO, BRUNO B.; ISAAC, LOURDES; BARBOSA, ANGELA S.; MORGANTI, LIGIA. Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3. Biotechnology Reports, v. 19, n. e00266, p. 1-7, 2018. DOI: 10.1016/j.btre.2018.e00266. Disponível em: http://repositorio.ipen.br/handle/123456789/29292. Acesso em: 30 Dec 2025.
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.