pTONA5: a hyperexpression vector in Streptomycetes |
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Authors: | Hatanaka Tadashi Onaka Hiroyasu Arima Jiro Uraji Misugi Uesugi Yoshiko Usuki Hirokazu Nishimoto Yukifumi Iwabuchi Masaki |
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Institution: | Research Institute for Biological Sciences, Okayama, Kaga-gun, Okayama, Japan. hatanaka@bio-ribs.com |
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Abstract: | We constructed the Streptomyces hyperexpression vector pTONA5 based on pIJ702 vector; it includes a metalloendopeptidase (SSMP) promoter isolated from Streptomyces cinnamoneus TH-2 and a metalloendopeptidase terminator isolated from Streptomyces aureofaciens TH-3. The vector contains recognition sites for restriction enzymes NdeI and EcoRI/XbaI/HindIII between the promoter and terminator to facilitate heterologous gene cloning. The plasmids were transferred from Escherichia coli to streptomycetes via conjugation from oriT; the transformants were able to be selected using kanamycin and/or thiostrepton. The SSMP promoter functions constitutively in the presence of a rich inorganic phosphate source and glucose. We constructed expression plasmids including three Streptomyces aminopeptidases—leucine aminopeptidase, proline aminopeptidase (PAP), and aminopeptidase P (APP)—using the pTONA5 vector and Streptomyces lividans. Although they lack signal peptides for secretion, PAP and APP were secreted at high levels in the culture broth. |
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Keywords: | Expression vector Conjugation Streptomycetes Aminopeptidase |
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