Immobilization of sugar-non-specific nucleases by utilizing the streptavidin--biotin interaction.
Due to their high enzymatic activity, the sugar-non-specific endonucleases from Serratia marcescens and Anabaena can be used for a number of applications, such as the removal of contaminating genetic material from biological preparations, footprinting studies, and the determination of nucleic acids in biochemical samples. These methods would benefit from immobilized nucleases. For this purpose, a single cysteine residue was added at the N-terminus of the Serratia and Anabaena nucleases and subsequently modified with a maleimide-biotin conjugate. Alternatively, a biotin acceptor domain was fused to the Anabaena nuclease, allowing biotinylation during expression in E. coli without a further chemical step. The attachment of biotin-modified nucleases to streptavidin-coated paramagnetic beads and to streptavidin-coated surface plasmon resonance sensor chips (to study interactions with substrate and inhibitor) worked well when aggregates present in the protein preparations were removed by ultrafiltration. These methods should be of general use for similar enzyme systems.
Gast
F U
FU
Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35292 Giessen, Germany. frank-ulrich.gast@gmd.de
Franke
I
I
Meiss
G
G
Pingoud
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002850:Chromatography, Gel / Q000379:methods; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017978:Receptors, Growth Factor / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014462:Ultrafiltration
D003062:Codon; D004800:Enzymes, Immobilized; D017978:Receptors, Growth Factor; C052136:biotin receptor; D001710:Biotin; D019809:Streptavidin; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D004722:Endoribonucleases; C085858:sugar-nonspecific nuclease; C021835:Serratia marcescens nuclease; D010727:Phosphoric Diester Hydrolases; D003545:Cysteine