Journal
METHODS
Volume 55, Issue 1, Pages 58-64Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2011.06.006
Keywords
Cell-free protein expression; Leishmania tarentolae; Overlap extension PCR; High throughput protein expression; Rab GTPase
Funding
- ARC [KA003363]
- University of Queensland
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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. (C) 2011 Elsevier Inc. All rights reserved.
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