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  Generation and Characterization of a Leishmania tarentolae Strain for Site-Directed in Vivo Biotinylation of Recombinant Proteins

Klatt, S., Hartl, D., Fauler, B., Gagoski, D., Castro-Obregon, S., & Konthur, Z. (2013). Generation and Characterization of a Leishmania tarentolae Strain for Site-Directed in Vivo Biotinylation of Recombinant Proteins. Journal of Proteome Research, 12(12), 5512-5519. doi:10.1021/Pr400406c.

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2013
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2013 American Chemical Society
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 Creators:
Klatt, S., Author
Hartl, D., Author
Fauler, B.1, Author              
Gagoski, D., Author
Castro-Obregon, S., Author
Konthur, Z., Author
Affiliations:
1Microscopy and Cryo-Electron Microscopy (Head: Thorsten Mielke), Scientific Service (Head: Christoph Krukenkamp), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479668              

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Free keywords: leishmania tarentolae in vivo biotinylation biotin ligase recombinant protein expression avi tag 2-d page lc-ms/ms transmission electron microscopy enzymatic biotinylation posttranslational modification expression system mammalian-cells biotin autophagy antibody vaccine vector target
 Abstract: Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme to express recombinant proteins with a His(6)-tag and, additionally, to site-specifically in vivo biotinylate them for detection. Biotinylation is a relatively rare modification of endogenous proteins that allows easy detection with negligible cross-reactivity. Here, we established a genetically engineered L. tarentolae strain constitutively expressing the codon-optimized biotin-protein ligase from Escherichia coli (BirA). We thoroughly analyzed the strain for functionality using 2-D polyacrylamide-gel electrophoresis (PAGE), mass spectrometry, and transmission electron microscopy (TEM). We could demonstrate that neither metabolic changes (growth rate) nor structural abnormalities (TEM) occurred. To our knowledge, we show the first 2-D PAGE analyses of L. tarentolae. Our results demonstrate the great benefit of the established L. tarentolae in vivo biotinylation strain for production of dual-tagged recombinant proteins. Additionally, 2-D PAGE and TEM results give insights into the biology of L. tarentolae, helping to better understand Leishmania species. Finally, we envisage that the system is transferable to human-pathogenic species.

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Language(s): eng - English
 Dates: 2013-04-302013-10-072013
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: Other: WOS:000328231300014
DOI: 10.1021/Pr400406c
ISSN: 1535-3893
 Degree: -

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Title: Journal of Proteome Research
  Other : J. Proteome Res.
Source Genre: Journal
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Publ. Info: Washington, D.C. : American Chemical Society
Pages: - Volume / Issue: 12 (12) Sequence Number: - Start / End Page: 5512 - 5519 Identifier: ISSN: 1535-3893
CoNE: https://pure.mpg.de/cone/journals/resource/111019664290000