title: Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.
abstract: Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.
authors.affiliation: Laboratory of Insect Pathology and Microbial Control, Faculty of Agriculture, Graduate School, Institute of Biological Control, Kyushu University, Fukuoka, Japan. iiyama@grt.kyushu-u.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iiyama
authors.affiliation: Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Jae Man
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Tuneyuki
authors.initials: T
authors.lastname: Tatsuke
authors.affiliation: Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mon
authors.affiliation: Laboratory of Insect Genome Science, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kusakabe
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics*; D012831:Bombyx / Q000821:virology*; D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D044090:Serratia liquefaciens / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003851:Deoxyribonucleases
keywords: Baculovirus-Bombyx mori protein expression system; Recombinant nuclease; Sec secretion pathway; Serratia liquefaciens; Signal peptide