Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system

Kovtun, Oleksiy, Mureev, Sergey, Johnston, Wayne A. and Alexandrov, Kirill (2010) Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system. PLoS One, 5 12: e14388-1-e14388-11. doi:10.1371/journal.pone.0014388


Author Kovtun, Oleksiy
Mureev, Sergey
Johnston, Wayne A.
Alexandrov, Kirill
Title Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system
Formatted title
Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2010-12-21
Sub-type Article (original research)
DOI 10.1371/journal.pone.0014388
Open Access Status DOI
Volume 5
Issue 12
Start page e14388-1
End page e14388-11
Total pages 11
Editor Damian Pattinson
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Collection year 2011
Language eng
Formatted abstract
The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.
Copyright: © 2010 Kovtun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keyword Protein
Evolution
Phosphatase
Disease
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Article # e14388

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
Institute for Molecular Bioscience - Publications
 
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Created: Thu, 24 Feb 2011, 00:22:49 EST by Susan Allen on behalf of Institute for Molecular Bioscience