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The LEXSY platform for recombinant protein expression

Reinhard BreitlingJena Bioscience GmbH, Jena, Germany; reinhard.breitling@jenabioscience.com

Due to the increasing demand for production of recombinant proteins in the proteomics era and the shortcomings of the traditional protein expression systems we have developed the LEXSY expression platform based on the protozoan organism Leishmania tarentolae. This unicellular eukaryotic host is safe (biosafety group S1), robust, easy and cost-efficient to culture and has the full capacity for eukaryotic protein folding and modification including mammalian-type post-translational glycosylation. We demonstrated an exceptionally homogeneous biantennary structure of fully galactosylated, core-α-1,6-fucosylated N-glycans of LEXSY expressed glycoproteins (Figure 1).

Based on the flexibility of the LEXSY technology, recombinant target proteins can be produced intracellularly or be secreted into the culture medium. Both, constitutive and inducible expression architectures are available.

Figure 1. Comparison of N-glycosylation pattern of glycoproteins produced in different protein expression systems. Glycosylation in LEXSY was investigated with human erythropoietin, human interferon gamma and host surface glycoprotein GP63. In all cases a biantennary, fully galactosylated, core-α-1,6-fucosylated N-glycan structure was found that is similar to mammalian-type glycosylation (Breitling et al., 2002).

. et al. (eds.), rm animal ,

DOI 10.3920/978-90-8686-776-9_ , © Wageningen Academic Publishers 2013

A de Almeida Fa proteomics 2013, Proceedings of the 4th Management Committee Meeting and 3rd Meeting of Working Groups 1, 2 & 3 of COST Action FA1002

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For constitutive expression, the target gene constructs are stably integrated into the chromosomal rDNA (ssu) locus and co-transcribed by the strong RNA polymerase I of the protozoan host cells (Breitling et al., 2002). For inducible expression, we established a L. tarentolae recipient strain co-expressing bacteriophage T7 RNA polymerase and tetracycline repressor. This strain was transfected with heterologous target genes placed under the control of a T7 promoter/TET-operator assembly, which allowed transcription initiation upon addition of tetracycline to the culture medium. The target gene constructs were either stably integrated into the chromosomal ß-tubulin (tub) or ornithine decarboxylase (odc) locus (Kushnir et al., 2005) or maintained episomaly (Kushnir et al., 2011). In a recent version induction of target protein expression can be monitored online during cultivation by a transcriptionally coupled fluorescence marker (www.jenabioscience.com).

Numerous proteins, including enzymes, surface antigens, toxins, antibodies and membrane proteins have been expressed with LEXSY (Table 1). Expression yields of up to several hundred mg per litre of culture were obtained, and the purified proteins were successfully employed for diagnostics and research and development including structure determination by NMR and X-ray crystallography (Niculae et al. 2006; Gazdag et al. 2010). LEXSY has recently been complemented by a version for in vitro translation for cell-free expression of recombinant proteins (Mureev et al., 2009, Kovtun et al., 2010).

Pilot plant cultivations in 30 litre bioreactor scale in standard bacteriological media revealed that LEXSY is fully adapted to fermentation technology. Target protein yields of >100 mg/l were obtained in high cell density fermentations (8×108 cells/ml) in suspension culture with this robust protozoan host.

In summary: LEXSY thus combines the advantages of a fast growing robust expression host with the full potential of an eukaryotic protein synthesis/folding/modification machinery and will contribute to meet the challenges of ongoing proteomics initiatives.

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Table 1. Typical examples of LEXSY-expressed proteins clustered by type of protein.1

Target protein Size (kDa) Yield (mg/l)

Cytoplasmic proteinsSOD1 16 30EGFP 28 300SPEE 35 30p85 of PI3 kinase 85 3smmyHC 154 1

Nuclear proteinsT7 RNA Pol 100 1

Secreted proteinsMHC II-b 30 500CRP 23 44SAG1&2 15/31 10Fc fusion 39 10MDP1 45 6Laminin 332 420 (150+135+135) 0.5

Membrane proteinsEGFP-Rab7 (mb-associated) 52 12BkrB2-GST (Type III TM7) 55 0.5PDM9 (Type I) 43 0.2

1 SOD1 = human Cu/Zn superoxide dismutase; EGFP = enhanced green fluorescent protein of A. victoria; SPEE = human spermidine synthetase; p85 = bovine Phosphoinositide 3-Kinase regulatory subunit α; smmyHC = heavy chain of human smooth muscle myosine; T7 RNA Pol. = RNA polymerase of phage T7 supplied with nuclear localization signal; MHC II-β = human Major Histocom-patibility Complex II β subunit; CRP = human C-reactive protein of pentaxin family; SAG1/2 = surface antigens of Toxoplasma gondii Fc fusion = N-terminal fusion of DNA binding domain to human Fc fragmant; MDP1 = human renal dipeptidase 1; Laminin 332 = large heterotrimeric human laminin glycoprotein α3β3γ2; EGFP-Rab7 = EGFP fusion of Ras-associated small GTP-binding protein Rab7 (membrane associated by prenylation); BrkB2-GST = GST fusion of human bradykinin receptor B2 (7TM transmembrane protein); PDM9 = human transmembrane protein with EGF-like and two follistatin-like domains 2 (type I membrane protein N out). For detailed description see www.jenabioscience.com.

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References

Breitling, R., Klingner, S., Callewaert, N., Pietrucha, R., Geyer, A., Ehrlich, G., Hartung, R., Müller, A., Contreras, R., Beverley, S.M. and Alexandrov, K. 2002. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expression and Purification 25, 209-218.

Gazdag, E.M., Cirstea, I., Breitling, R., Lukes, J., Blankenfeldt, W. and Alexandrov, K., 2010. Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae. Acta Crystallographica F66, 871-877.

Kovtun, O., Mureev, S., Johnston, W. and Alexandrov, K., 2010. Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System. PLOS one 5, e14388, doi: http://dx.doi.org/10.1371/journal.pone.0014388.

Kushnir, S., Cirstea, I., Basiliya, L., Lupilova, N., Breitling, R. and Alexandrov, K., 2011. Artificial linear episome-based protein expression system for protozoon Leishmania tarentolae. Molecular & Biochemical Parasitology 176, 69-79.

Kushnir, S., Gase, K., Breitling, R. and Alexandrov, K., 2005. Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expression and Purification 42, 37-46.

Mureev, S., Kovtun, O., Nguyen, U.T.T. and Alexandrov, K., 2009. Species-independent translational leaders facilitate cell-free expression. Nature Biotechnology 27, 747-752.

Niculae, A., Bayer, P., Cirstea, I., Bergbrede, T., Pietrucha R., Gruen, M., Breitling, R. and Alexandrov, K., 2006. Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae. Protein Expression and Purification 48, 167-172.