Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling.
In murine models of Crohn's disease, rheumatoid arthritis and colon cancer, IL-6 (interleukin-6) signalling via the sIL-6R (soluble IL-6 receptor; termed IL-6 trans-signalling) has been shown to promote the pathology associated with these conditions. These detrimental activities can, however, be selectively blocked by soluble forms of the gp130 (glycoprotein 130) receptor. Although sgp130 (soluble gp130) therefore represents a viable therapeutic modality for the treatment of these conditions, the mass manufacture of such biologics is often expensive. The advent of molecular farming has, however, provided an extremely cost-effective strategy for the engineering of recombinant proteins. Here, we describe the expression and production of a biologically active sgp130 variant that is expressed in transgenic tobacco plants as an ELP (elastin-like peptide)-fusion protein (mini-gp130-ELP). Mini-gp130-ELP consists of the first three domains of gp130 (Ig-like domain and cytokine binding module) fused to 100 repeats of ELP. Expression of mini-gp130-ELP did not affect the growth rate or morphology of the transgenic plants, and purification was achieved using inverse transition cycling. This approach led to an overall yield of 141 microg of purified protein per g of fresh leaf weight. The purified mini-gp130-ELP specifically inhibited sIL-6R-mediated trans-signalling as measured by binding to the IL-6-sIL-6R complex and through its ability to block sIL-6R-mediated activation of STAT3 (signal transducer and activator of transcription 3) phosphorylation and proliferation in human hepatoma cells and murine pre-B-cells. Consequently, the present study validates the potential application of molecular farming in transgenic tobacco plants as a strategy for the expression and purification of therapeutically advantageous biologics such as sgp130.
Lin
Meng
M
Biochemisches Institut, Christian-Albrechts Universität zu Kiel, Olshausenstr. 40, D-24098 Kiel, Germany.
Rose-John
Stefan
S
Grötzinger
Joachim
J
Conrad
Udo
U
Scheller
Jürgen
J
D000818:Animals; D002460:Cell Line; D050822:Cytokine Receptor gp130 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism*
D015850:Interleukin-6; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130