title: Leishmania cell-free protein expression system.
abstract: Cell-free protein expression is an important tool for a rapid production, engineering and labeling of recombinant proteins. However the complex protocols for preparation of eukaryotic cell-free protein expression systems result in high manufacturing costs and limit their utility. Recently we reported a novel cell-free expression system based on the lysate of a fermentable protozoan Leishmania tarentolae. Herein we describe a protocol for high throughput protein expression using Leishmania cell-free lysate. The protocol combines PCR-based synthesis and engineering of translation templates with a combined transcription-translation system. The protocol is adapted to multiwell plate format and allows translation of large protein libraries. In the presented example we translate in vitro and isolate a nearly complete complement of mammalian Rab GTPases. Further applications and developments of the system are discussed.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Oleksiy
authors.initials: O
authors.lastname: Kovtun
authors.forename: Sergey
authors.initials: S
authors.lastname: Mureev
authors.forename: WooRam
authors.initials: W
authors.lastname: Jung
authors.forename: Marta H
authors.initials: MH
authors.lastname: Kubala
authors.forename: Wayne
authors.initials: W
authors.lastname: Johnston
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D007891:Leishmania / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008322:Mammals; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins