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

A series of T7-promoter based bicistronic expression vectors was constructe d 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 forma tion of NucA, independent of the order of the genes, the relative expressio n of the two proteins and the temperature applied during expression. Two co nstructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared wit h nucA. In these cells inclusion bodies are formed which contain NucA and N uiA in a 1:1 molar ratio. The complex can be solubilized with 6 M urea afte r 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, ou r system leads to a highly pure and homogeneous complex preparation, as req uired for biophysical and structural studies. Thus, our new method is a sup erior alternative for the production of the NucA/NuiA complex in which sepa rately produced nuclease and inhibitor are mixed, and an excess of one or t he other component, as well as aggregates of NucA, have to be removed from the preparation. (C) 2000 Elsevier Science B.V. All rights reserved.