Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, albeit with weak immunogenicity. In this study, the xenoantigen l-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via the anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity toward A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancers of tumor associated antigens in the development of tumor immunotherapies.

Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45 °C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.Graphical abstractHighlights► Galactokinase from Streptococcus pneumoniae TIGR4 has wide sugar substrate specificity. ► GalKSpe4 can recognize glucose as substrate. ► Most interestingly, GalKSpe4 exhibited activity for GalNAc. ► This is the first time to report that GALK in bacteria can recognize GalNAc.

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.

Interferon-β has anti-viral, anti-proliferation and multifunctional immunomodulatory activities and shows promising clinical effects for treatment of inflammatory disorders. The recombinant human interferon-β (huIFN-β) 1b was expressed in the food-grade lactic acid bacterium, Lactococcus lactis, using a nisin-controlled gene expression system. huIFN-β production from recombinant strains (with and without LEISSTCDA propeptide) was approximately 21 and 7 μg l−1, respectively. Moreover, 95% (former strain) and 88% (latter strain) of total recombinant proteins were secreted into the culture medium. The biological activities of huIFN-β from recombinant strains revealed similar antiviral activities of 107 I.U. mg−1. These results demonstrate the potential application of recombinant strains as a food grade vehicle to deliver bioactive huIFN-β in vivo.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharide in a(n) (oligo)saccharide mixture. The single terminal aldehydes of oligoglucoside residues released by acid hydrolysis of β-1, 3-d-glucan from yeast were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligoglucoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligoglucoside moieties in heterogeneous sample. Therefore, FACE represents an accessible, sensitive, and quantitative analytical tool enabling the analysis of (oligo)saccharides derived from different sources.

A versatile vector was developed for heterologous proteins display on the cell surface of Pichia pastoris using the C-terminal half of α-agglutinin from Saccharomyces cerevisiae as a membrane anchor under the control of the alcohol oxidase 1 promoter (pAOX1). Multiple cloning sites and the sequence encoding the Xpress epitope (-Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys-) were introduced into the vector for insertion of heterologous genes and selective cleavage of target proteins. Enhanced green fluorescence protein (EGFP) was used as a model protein to check the function of this vector. The expression of EGFP on the P. pastoris surface was confirmed by confocal laser scanning microscopy. Fluorescence microscopy and western blot analysis confirmed that EGFP can be successfully cleaved from the cell surface by treating with enterokinase.

The nodC gene from Mesorhizobium loti was cloned into E. coli, leading to production of chitin oligosaccharides (COs)—mainly penta-N-acetyl-chitopentaose. A two-step fermentation procedure was then developed which gave 930 mg CO/L with a productivity of 37 mg/l·h.