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 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 6M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2mg of the complex are obtained from 1l 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.
Institut für Biochemie, Fachbereich Biologie, Chemie und Geowissenschaften, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392, Giessen, Germany.
C
C
Korn
G
G
Meiss
F
F
Gast
O
O
Gimadutdinow
C
C
Urbanke
A
A
Pingoud
D020418:Algal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017033:Anabaena / Q000201:enzymology / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004720:Endonucleases; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010726:Phosphodiesterase Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D014461:Ultracentrifugation
D020418:Algal Proteins; D046911:Macromolecular Substances; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D004720:Endonucleases; C085858:sugar-nonspecific nuclease; D010727:Phosphoric Diester Hydrolases