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Genetic engineering of Escherichia coli to produce a 1:1 complex of the Anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA

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dc.contributor.author Korn C.
dc.contributor.author Meiss G.
dc.contributor.author Gast F.
dc.contributor.author Gimadutdinow O.
dc.contributor.author Urbanke C.
dc.contributor.author Pingoud A.
dc.date.accessioned 2018-09-17T20:43:15Z
dc.date.available 2018-09-17T20:43:15Z
dc.date.issued 2000
dc.identifier.issn 0378-1119
dc.identifier.uri https://dspace.kpfu.ru/xmlui/handle/net/133939
dc.description.abstract A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp. PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA. With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression. Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA. In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio. The complex can be solubilized with 6 M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2 mg of the complex are obtained from 1:1 Escherichia coli culture. As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies. Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation. (C) 2000 Elsevier Science B.V.
dc.relation.ispartofseries Gene
dc.subject Complex formation
dc.subject Copurification
dc.subject Tandem expression
dc.title Genetic engineering of Escherichia coli to produce a 1:1 complex of the Anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA
dc.type Article
dc.relation.ispartofseries-issue 2
dc.relation.ispartofseries-volume 253
dc.collection Публикации сотрудников КФУ
dc.relation.startpage 221
dc.source.id SCOPUS03781119-2000-253-2-SID0034622560


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  • Публикации сотрудников КФУ Scopus [24551]
    Коллекция содержит публикации сотрудников Казанского федерального (до 2010 года Казанского государственного) университета, проиндексированные в БД Scopus, начиная с 1970г.

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