Expression of single-chain variable fragments fused with the Fc-region of rabbit IgG in Leishmania tarentolae.

BACKGROUND: In recent years the generation of antibodies by recombinant methods, such as phage display technology, has increased the speed by which antibodies can be obtained. However, in some cases when recombinant antibodies have to be validated, expression in E. coli can be problematic. This primarily occurs when codon usage or protein folding of specific antibody fragments is incompatible with the E. coli translation and folding machinery, for instance when recombinant antibody formats that include the Fc-region are needed. In such cases other expression systems can be used, including the protozoan parasite Leishmania tarentolae (L. tarentolae). This novel host for recombinant protein expression has recently shown promising properties for the expression of single-chain antibody fragments. We have utilised the L. tarentolae T7-TR system to achieve expression and secretion of two scFvs fused to the Fc-region of rabbit immunoglobulin G (IgG). RESULTS: Based on the commercial vector pLEXSY_IE-blecherry4 (Jena Bioscience; Cat. No. EGE-255), we generated a vector containing the Fragment Crystallisable (Fc) region of rabbit IgG allowing insertions of single chain antibody fragments (scFvs) in frame via Ncol/Notl cloning (pMJ_LEXSY-rFc). For the expression of rabbit Fc-fusion scFvs (scFv-rFc) we cloned two scFvs binding to human vimentin (LOB7 scFv) and murine laminin (A10 scFv) respectively, into the modified vector. The LOB7-rFc and A10-rFc fusions expressed at levels up to 2.95 mg/L in L. tarentolae T7-TR. Both scFv-rFcs were purified from the culture supernatants using protein A affinity chromatography. Additionally, we expressed three different scFvs without the rFc regions using a similar expression cassette, obtaining yields up to 1.00 mg/L. CONCLUSIONS: To our knowledge, this is the first time that antibody fragments with intact Fc-region of immunoglobulin have been produced in L. tarentolae. Using the plasmid pMJ_LEXSY-rFc, L. tarentolae T7-TR can be applied as an efficient tool for expression of rFc fusion antibody fragments, allowing easy purification from the growth medium. This system provides an alternative in cases where antibody constructs express poorly in standard prokaryotic systems. Furthermore, in cases where bivalent Fc-fused antibody constructs are needed, using L. tarentolae for expression provides an efficient alternative to mammalian expression.

Rayleigh Instability-Driven Coaxial Spinning of Knotted Cell-Laden Alginate Fibers as Artificial Lymph Vessels.

Constructing artificial lymph vessels, which play a key role in the immune response, can provide new insights into immunology and disease pathologies. An immune tissue is a highly complex network that consists of lymph vessels, with a "beads-on-a-string" knotted structure. Herein, we present the facile and rapid fabrication of beads-on-a-string knotted cell-laden fibers using coaxial spinning of alginate by exploiting the Plateau-Rayleigh instability. It is shown how alterations in the flow rate and alginate concentration greatly affect the beads-on-a-string structure, rooted in the Plateau-Rayleigh instability theory. Biocompatibility was confirmed by the lactate dehydrogenase (LDH) assay and live/dead staining of the encapsulated human white blood cells. Finally, the encapsulated white blood cells were still functional as indicated by their anti-CD3 activation to secrete interleukin 2. The rapid fabrication of a cell-laden beads-on-a-string three-dimensional (3D) culture platform enables a crude mimicry of the lymph vessel structure. With joint expertise in immunology, microfluidics, and bioreactors, the technology may contribute to the mechanistic assay of human immune response in vitro and functional replacement.

3D anisotropic photocatalytic architectures as bioactive nerve guidance conduits for peripheral neural regeneration.

Great research efforts have been invested in developing nerve guidance conduits (NGCs), which can direct axons advance and guide peripheral neural regeneration. Here, three different aspects of NGC design, namely anisotropy, photocatalytic stimulation and self-assembly at implantation site, were unitedly addressed. Firstly, melt electrowriting (MEW) was used to print anisotropic, microfibrous PCL architectures. Specifically, by tailoring the fiber spacing ratio between two arms of the grid patterns (1-1, 1-2, 1-3), preferential neurite extension of PC 12 cells along the long arm direction was achieved. Such anisotropic neurites guidance was further strengthened when the intersection angles were reduced from 90° to 30°. Secondly, functionalization of PCL micropatterns with graphene oxide and graphitic carbon nitride (g-C3N4), a visible-light photocatalyst, may enable optoelectronic conversion and wireless neural stimulation. As a result, photocatalytic stimulation further enhanced neurite extension length under visible light irradiation. Last but not the least, NGC were successfully obtained either by manually rolling or self-assembly using a thermo-responsive bi-layer system. Interestingly, the anisotropic micropattern design dictated the self-assembly process, and an underlying mechanism was proposed. With a synergy of three unique design parameters, the herein presented NGCs may possess great potential for repairing peripheral nerve injuries.

An anti vimentin antibody promotes tube formation.

In recent years, there has been an increasing appreciation of the importance of secreted and extracellular proteins that traditionally have been considered as intracellular components. Vimentin is a highly abundant intermediate filament protein, and its intracellular functions have been investigated in a large number of studies. Recently, however, vimentin has been shown to take part in significant processes outside the cell. Our understanding of the functions of extracellular vimentin is, however, limited. In this study we demonstrate that a vimentin specific antibody, obtained by phage antibody technology, promotes tube formation of endothelial cells in a 2D matrigel assay. By binding vimentin, the antibody increases the tube formation by 21% after 5 hours of incubation. Addition of the antibody directly to cultured endothelial cells does not influence endothelial cell migration or proliferation. The enhanced tube formation can be seen for up to 10 hours where after the effect decreases. It is shown that the antibody-binding site is located on the coil 2 domain of vimentin. To our knowledge this is the first study that demonstrates an enhanced tube formation by binding vimentin in a 2D matrigel assay under normoxic conditions.