Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 62, Issue 2, Pages 244-248Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.09.001
Keywords
Expression vector; Conjugation; Streptomycetes; Aminopeptidase
Categories
Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan
- New Energy and Industrial Technology Development Organization (NEDO) of Japan
Ask authors/readers for more resources
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. (c) 2008 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available