Cloning, expression and purification of recombinant nucleases. (A) A ClustalW alignment of the SP I amino acid sequence with homologous sequences. Amino acid numbering is given with respect to the primary structure of mature P1. P1 nuclease of Penicillium citrinum [GenBank:P24289]; S1 nuclease of Aspergillus oryzae [GenBank:AAB20216]; M1 nuclease of Mesorhizobium loti [GenBank:BAB52626]; BEN1 nuclease of Hordeum vulgare [GenBank:BAA28942]; CEL I nuclease of Apium graveolens [GenBank:AAF42954]; SP I nuclease of Spinacia oleracea [GenBank:ABK34453]. The nucleotide binding sequence of P1 is underlined . Symbols: *, identity; :, strong similarity; ., weak similarity; ^, resudies identical in CEL I and SP I. (B) Detection of single-strand DNase activities after in-gel enzyme refolding. Lane 1 and 2, Ni2+ affinity-purified SP Iwt and SP IH135A nucleases, respectively; lanes 3 and 5, native CEL nuclease purified from celery, after the MonoQ step; this sample is a combination of CEL I and CEL II nucleases ; lane 4, recombinant CEL I nuclease purified on a Ni2+ affinity column. (C) Induction of single-strand specific activity in infected Sf9 cells detected by RF-I nicking assay. Lanes 1–4, 0.1 μl cell extract was used in 20 μl reaction; lanes 7–9, 1 μl cell culture media in 20 μl reaction; lanes 1 and 7, CEL I nuclease expression; lanes 2 and 8, SP Iwt nuclease expression; lanes 3 and 9, cells infected with an "empty" control vector containing no nuclease gene; lane 4, extract of non-infected cells; lane 5, native CEL nuclease purified from celery; lane 6, uncut pUC19 DNA. I, RF-I supercoiled plasmid DNA; II, RF-II nicked circular plasmid DNA; III, RF-III linearized plasmid DNA.