Bio-functional enhancement of plant based-haemagglutinin by fusion with IgMFc

The creation of recombinant oligomeric haemagglutinin proteins from A/H5N1 virus is a new concern for many scientists. In this study, the gene encoding the haemagglutinin protein (H5TG) derived from virus A/duck/Vietnam/TG24-01/2005 was fused with three different motifs (GCN4pII, GCN4pII-IgMFc and GCN4pII-ELP-IgMFc) in order to form three recombinant proteins (trimeric H5TGpII, oligomeric H5TGpII-IgMFc and ELPylated oligomeric H5TGpII-ELP-IgMFc, respectively) for enhancing protein expression, bio-function and purification of H5TG. These H5TG fragments have been attached to expression cassettes in the pCB301 shuttle vector, transiently expressed in Nicotiana benthamiana by agroinfiltration, then the protein expression was confirmed by SDS-PAGE and Western blot analysis. The results showed that the expression level of the H5TGpII-ELP-IgMFc protein in plant raw extract was stronger than that of two other proteins analyzed in this paper. Evaluation of bio-function showed that the haemagglutination titre (HA titre) of the total solution protein extract containing H5TGpII-IgMFc protein was highest (64 HAU) compared to that containing two remaining proteins (8 HAU). Subsequently, the H5TGpII and H5TGpII-IgMFc proteins were purified by immobilized metal ion affinity chromatography (IMAC), while the H5TGpII-ELP-IgMFc protein was purified using membrane-based Inverse Transition Cycling (mITC). The oligomer state of the purified proteins was then determined by non-reducing SDS-PAGE. The haemagglutination assay analysis of purified proteins showed that the lowest protein amount causing erythrocyte agglutination (1 HAU) of the H5TGpII-IgMFc protein was 0.06 µg and lower four times than that of H5TGpII and H5TGpII-ELP-IgMFc proteins (both of them were 0.24 µg). This indicates that the fusing of the GCN4pII-IgMFc motif into the H5TG protein gives the stronger bio-function than the fusing of two remaining motifs into this protein. This result opens up the applicability of the IgMFc for generation of oligomer proteins to enhance the bio-function of target proteins in the study of recombinant vaccine production.

Elastin-Like Polypeptides as Thermosensitive Polymer System

Thermo-responsive elastin-like polypeptides (ELPs) were successfully obtained by inverse transition cycling (ITC) and recursive directional ligation (RDL). Six ELPs displayed thermal properties, depending on their sequence and chain length. It was found that the ELP[KV8F-4 and ELP[KV8F-8 were effective as thermosensitive materials at the body temperature with phase transition temperature from 35 to 45oC.

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 μg 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.