Species-independent translational leaders facilitate cell-free expression

Abstract

Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems differ substantially from each other with respect to efficiency, scalability and the ability to produce functional eukaryotic proteins. Here we describe species-independent translational sequences (SITS) that mediate efficient cell-free protein synthesis in multiple prokaryotic and eukaryotic systems, presumably through bypassing the early translation initiation factors. We use these leaders in combination with targeted suppression of the endogenous Leishmania tarentolae mRNAs to create a cell-free system based on this protozoan. The system can be directly programmed with unpurified PCR products, enabling rapid generation of large protein libraries and protein variants. L. tarentolae extract can produce up to 300 microg/ml of recombinant protein in 2 h. We further demonstrate that protein-protein and protein-small molecule interactions can be quantitatively analyzed directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy.

Cortactin Scaffolds Arp2/3 and WAVE2 at the Epithelial Zonula Adherens

“…Proteins of interest were cloned into a derivative of the pLTE vector carrying T7 promoter and species-independent translation initiation sequences as well as His or fluorescent protein tags (22). The vectors included recombination sites compatible with Gateway cloning system (Clontech).…”

“…For these experiments, we chose to use a eukaryotic cell-free translation system based on Leishmania tarentolae extract (LTE). LTE is well suited for the analysis of protein interactions as it supports co-expression of multiple cDNAs and allows production of large multidomain proteins in their active form (22,29). We were able to express full-length polypeptides of EcadTail, cortactin, or ␤-catenin tagged with either GFP or mCherry in the LTE system.…”

Development of an artificial cell, from self-organization to computation and self-reproduction

“…Rather, genetic modules work with specific transcription factors and their respective DNA promoters and operators. The recent preparation of a universal T7 transcription system extends the possibilities to express genes from various organisms, but it does not solve for the lack of modularity (40). Compared to bacteriophage transcription, the structure of bacterium promoters allows for a much larger modularity of transcription, even with a single transcription factor.…”

Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A) – dependent intracellular trafficking through recycling endosomes

“…A mCherry-His or His tag was fused with the ORFs at the C-terminus using a short linker region TCCGGTTCGGGCTCCGGTGGA. Poly(U) species independent translation enhancers (SITS) were used as a translation initiator [16] and combined with ORF-coding fragments together with the mCherry-His-coding fragment by overlap-extension PCR (OE-PCR) [13]. Templates for the expression of N-terminally labeled bait proteins were assembled by combining amplified poly(U)SITS-EGFP DNA fragment and a protein coding fragment.…”

“…Leishmania tarentolae cell-free system -To validate identified interactors, we recombinantly produced them in the cell-free system based on a protozoan Leishmania tarentolae [16]. In vitro translation reactions were primed with DNA templates coding for FTase and RabGGTase α subunits and the potential interacting proteins using a previously described protocol [13].…”