Hydrophobic Tagging Research Articles

Low Micromolar Inhibitors of Galectin-3 Based on 3′-Derivatization of N-Acetyllactosamine

A strategy for generating potential galectin inhibitors was devised based on derivatization at the C-3' atom in 3'-amino-N-acetyllactosamine by using structural knowledge of the galectin carbohydrate recognition site. A collection of 12 compounds was prepared by N-acylations or N-sulfonylations. Hydrophobic tagging of the O-3 atom in the N-acetylglucosamine residue with a stearic ester allowed rapid and simple product purification. The compounds were screened in a galectin-3 binding assay and three compounds with significantly higher inhibitory activities compared to the parent N-acetyllactosaminide were found. These three best inhibitors all carried an aromatic amide at the C-3' position of the galactose moiety, which indicates that favorable interactions were formed between the aromatic group and galectin-3. The best inhibitor had an IC50 value (4.4 microM) about 50 times better than the parent N-acetyllactosaminide, which implies that it has potential as a valuable tool for studying galectin-3 biological functions and also as a lead compound for the development of galectin-3-blocking pharmaceuticals.

Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation

We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce M5-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.