The amino acidic substitution of cysteine 167 by serine (C<SUP>167</SUP>S) in <i>Bst</i>VI restriction endonuclease of <i>Bacillus stearothermophilus</i> V affects its conformation and thermostability

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dc.contributor.authorLoyola, C
dc.contributor.authorSaavedra, C
dc.contributor.authorGómez, I
dc.contributor.authorVásquez, C
dc.date.accessioned2025-01-21T01:32:11Z
dc.date.available2025-01-21T01:32:11Z
dc.date.issued1999
dc.description.abstractThe restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation (CS)-S-134 was purified to homogeneity but appeared to be very unstable. In contrast, the (CS)-S-167 mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the (CS)-S-167 BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease. (C) Societe francaise de biochimie et biologie moleculaire / Elsevier, Paris.
dc.fuente.origenWOS
dc.identifier.issn0300-9084
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/97219
dc.identifier.wosidWOS:000080524500010
dc.issue.numero3
dc.language.isoen
dc.pagina.final266
dc.pagina.inicio261
dc.revistaBiochimie
dc.rightsacceso restringido
dc.subjectrestriction endonuclease
dc.subjectrecombinant PCR
dc.subjectsite-directed mutagenesis
dc.subjectB-stearothermophilus
dc.subject.ods03 Good Health and Well-being
dc.subject.odspa03 Salud y bienestar
dc.titleThe amino acidic substitution of cysteine 167 by serine (C<SUP>167</SUP>S) in <i>Bst</i>VI restriction endonuclease of <i>Bacillus stearothermophilus</i> V affects its conformation and thermostability
dc.typeartículo
dc.volumen81
sipa.indexWOS
sipa.trazabilidadWOS;2025-01-12
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