title: Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system.
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.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Oleksiy
authors.initials: O
authors.lastname: Kovtun
authors.forename: Sergey
authors.initials: S
authors.lastname: Mureev
authors.forename: Wayne
authors.initials: W
authors.lastname: Johnston
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D002474:Cell-Free System / Q000378:metabolism; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007891:Leishmania / Q000378:metabolism*; D008954:Models, Biological; D008957:Models, Genetic; D008967:Molecular Biology / Q000379:methods*; D010963:Plasmodium falciparum / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods; D017510:Protein Folding; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism
chemical_list: D020543:Proteome; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020691:rab GTP-Binding Proteins