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* In this issue:

(1) NEW: LEXSY In Vitro Translation Kits

  • For high yields: Plasmid-based In Vitro LEXSY
  • For rapid results: PCR-based In Vitro LEXSY
(2) Selected examples

   
       
  
   
   

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    (1) NEW: LEXSY In Vitro Translation Kits
   


In vitro (cell-free) expression of recombinant proteins has become a powerful method for production of recombinant proteins. It plays a central role in a wide variety of applications such as functional analysis and biochemical characterization of proteins and protein interactions, investigation of protein translation mechanisms, protein engineering, in vitro evolution, and structural biology (Katzen et al. 2005). Its multiplexed format can be used for production of protein arrays for many applications including drug screening and diagnostics (He et al. 2007).

The main advantages of cell-free protein expression are its rapidity of only few hours and its independence of living host-organisms. These features enable very fast generation of results and greatly alleviate typical expression problems due to toxicity and/or degradation of the protein of interest.

Our LEXSY In Vitro Translation System is a NEW, rapid, convenient, flexible and cost-efficient tool for production of recombinant proteins based on the cell extract of the protozoon Leishmania tarentolae (Mureev et al. 2009).

In contrast to the established E. coli in vitro translation, LEXSY contains chaperones for correct folding of proteins of higher eukaryotes and is capable of performing the full set of post-translational modifications (Kovtun et al. 2010). Further, compared to the insect, rabbit and wheat germ systems, LEXSY yielded significant higher expression levels (Figure 1A). Finally, our LEXSY In Vitro Translation System allows efficient suppression of background translation without addition of e.g. nucleases that are often required in other cell-free systems. Instead, an anti-splice leader oligonucleotide blocks translation of endogenous mRNA making use of the unique genetic organization of the LEXSY host.

Dependent on the way of template preparation two versions are available:

  • For high yields: Plasmid-based In Vitro LEXSY

    The Plasmid-based LEXSY In Vitro Translation is recommended for high-yield and/or large volume reactions. It is also recommended for open reading frames larger than 2500 bp and requires sub-cloning of the target ORF into the pLEXSY_invitro-2 vector provided in the plasmid-based kit (Cat.-No. EGE-2002-15).

      View Manual (PDF, 2.4 MB)

  • For rapid results: PCR-based In Vitro LEXSY

    The PCR-based LEXSY In Vitro Translation is rapid and flexible. It utilizes PCR-mediated fusion of the target ORF to a T7 promoter and leader sequence by overlap extension (OE) technique and does not require any cloning step. Therefore, this approach allows rapid generation of large protein libraries directly from unpurified PCR products.

      View Manual (PDF, 2.4 MB)


   
    (2) Selected Examples
   


Figures 1B-D show examples of proteins produced with LEXSY In Vitro Translation Kits. Enhanced Green Fluorescent Protein (EGFP) and its fusion proteins can conveniently be detected directly in SDS-PAGE gels by in situ fluorescence scanning (Figures 1B and C) or isolated by affinity chromatography on GFP-Cap matrix for subsequent detection by conventional Coomassie staining (Figure 1D). For non-fluorescent protein targets Western blotting can be used for visualization.

Figure 1

A: Coupled transcription-translation of PCR generated EGFP template in LEXSY compared to other commercially available in vitro translation systems. The EGFP ORF was amplified by overlap-extension PCR and fused individually with the translational leaders according to the instructions of the cell-free systems manufactures. For details refer to Mureev et al. 2009.

B: Triplicate analysis of cell-free production of EGFP reference protein with plasmid-based (lanes 1-3) and PCR-based (lanes 4-6) In Vitro LEXSY. (Lane 7: negative no-template control, MW: molecular weigt marker). The in vitro reactions were carried out for 2 h at 20°C, resolved on 12% SDS-PAGE and in situ visualized on a UV transilluminator. Expression levels of the plasmid-based method (lanes 1-3) are approximately 2-fold higher than those of the PCR-based method.

C: Simultaneous in vitro co-expression of four proteins (EGFP and three EGFP fusion proteins, Rab7 = Ras-related small GTPase7, SOD = Cu/Zn superoxide dismutase, GST = Glutathione-S-Transferase) in a single extract. The in vitro reactions were resolved on SDS-PAGE and the products detected by in situ fluorescence. (Adapted from Mureev et al. 2009). All proteins are present at similar yields indicating the suitability of the system for production of heteromeric protein complexes.

D: EGFP-Cap matrix purification of in vitro produced EGFP fusion proteins. 1 = Rab8 (Ras-related small GTPase8)-EGFP, 2 = Cog5 (Complex of Golgi5)-EGFP, 3 = Cog8 (Complex of Golgi8)-EGFP, 4 = Rab1 (Ras-related small GTPase1)-EGFP, 5 = RabGGTβ (Rab Geranyl-Geranyl Transferase β)-EGFP, 6 = MBP (Maltose Binding Protein)-EGFP, 7 = EGFP. In vitro reactions and GFP-Cap matrix purification were performed as described in the In Vitro LEXSY user manual. The purified target proteins were resolved by SDS-PAGE and Coomassie stained. Right lane, molecular size protein marker (kDa). Adapted from Kovtun et al. 2010.


Find more details on In Vitro LEXSY cell free protein production here:
  Mureev et al. (2009) Species-independent translational leaders facilitate cell-free expression. Nature Biotechnology 27:747. (PDF, 2.2 MB)

  Kovtun et al. (2010) Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System. PLOS one 5:e14388. (PDF, 1.2 MB)


Cited References:
He et al. (2007) Arraying proteins by cell-free synthesis. Biomol. Eng. 24:375.

Katzen et al. (2005) The past, present and future of cell-free protein synthesis. Trends Biotechnol. 23:150.


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