Isolation of a new human scFv antibody recognizing a cell surface binding site to CEACAM1. Large yield production, purification and characterization in E. coli expression system.

The CEACAM1 cell adhesion molecule has recently received considerable interest as a tumour target antigen since its re-expression often occurs in the advanced stages of multiple malignancies including malignant melanoma, non-small cell lung cancer and other types of solid tumors. In this study, we describe the expression-purification and characterization of the new single chain variable fragment (scFv) antibody named DIATHIS1, that recognizes the N-terminal IgV-like domain present in CEACAM1. Three validation batches show that the production process is robust and reproducible. The scFv DIATHIS1 is formulated as a naturally occurring mixture of monomer and dimer. The antibody is biophysically stable at low temperature (-80°C), different concentrations and remains biologically active for at least 24months. The thermal stability of scFv DIATHIS1 at 37°C shows important features for its activity in vivo. The dimer behaves as a reservoir converting slowly into monomer. The monomer and dimer forms of scFv DIATHIS1 were isolated and characterized, showing high reactivity for CEACAM1. This new composition of antibody could have advantageous pharmacokinetics parameters over conventional scFv for in vivo applications.

Isolation of a novel gene from Photobacterium damselae subsp. piscicida and analysis of the recombinant antigen as promising vaccine candidate.

Photobacterium damselae subsp. piscicida (PDP) is the causative agent of fish pasteurellosis, a bacterial disease causing important losses in marine aquaculture. Vaccines against the pathogen can be a way to control the infection and avoid antibiotic treatments. However, a satisfactory protective vaccine against fish pasteurellosis is not commercially available. In this study, a biotechnogical approach based on reverse vaccinology has been used to identify potential vaccine candidates for the development of a recombinant subunit vaccine. Genome sequencing of clones from a genomic cosmid library of PDP and in silico selection of the surface exposed proteins were the initial steps in vaccine candidate identification. From 370 open reading frames (ORF) eight potential antigens were selected, expressed as recombinant proteins and purified. These vaccine candidates were used to generate specific polyclonal antibodies in mice. Each antibody was then screened in vitro by inhibition adherence assay of live PDP on chinook salmon embryo cells (CHSE-214). A lipoprotein, found to be involved in the adherence of the bacterium to epithelial cells and annotated as PDP_0080, was then selected. The recombinant protein was further investigated in fish vaccination and challenge experiments to assess its ability to protect sea bass, Dicentrarchus labrax, against PDP infection. Immunisation with PDP_0080 recombinant protein elicited high specific antibody titres. Furthermore, the survival rate of fish immunized with the 25 µg dose of protein was significantly higher compared to the control group. The results of the study suggest that the PDP_0080 protein could be a promising candidate for the design of a recombinant vaccine against pasteurellosis.

Immunophilin-loaded erythrocytes as a new delivery strategy for immunosuppressive drugs.

Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5±1.3, 7.5±3.1 and 15.5±0.4nmol FKBP12 were able to bind 3.5±1.5, 6.0±1.9 and 11.4±2.9µg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0±0.6, 5.0±0.8 and 15.9±2.4nmol of cyclophilin A could bind 8.9±3.4, 12.2±3.5 and 17.0±3.2µg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0±0.3µg FK506 and 3.2±0.3µg CsA). Moreover, FK506 released from FKBP12-RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking.

The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.

Expression, purification, and characterization of the recombinant putative periplasmic hemin-binding protein (HutB) of Photobacterium damselae subsp. piscicida.

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K(d)) of 10 microM.

Bovine hexokinase type I: full-length cDNA sequence and characterisation of the recombinant enzyme.

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain.