Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins

Klatt, Stephan, Hartl, Daniela, Fauler, Beatrix, Gagoski, Dejan, Castro-Obregon, Susana and Konthur, Zoltan (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


Author Klatt, Stephan
Hartl, Daniela
Fauler, Beatrix
Gagoski, Dejan
Castro-Obregon, Susana
Konthur, Zoltan
Title Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins
Formatted title
Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins
Journal name Journal of Proteome Research   Check publisher's open access policy
ISSN 1535-3893
1535-3907
Publication date 2013-12-01
Sub-type Article (original research)
DOI 10.1021/pr400406c
Open Access Status Not yet assessed
Volume 12
Issue 12
Start page 5512
End page 5519
Total pages 8
Place of publication Washington, DC, United States
Publisher American Chemical Society
Language eng
Formatted 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 His6-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.
Keyword 2-D PAGE
AVI tag
Biotin ligase
In vivo biotinylation
LC-MS/MS
Leishmania tarentolae
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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