Cell-free protein-based enzyme discovery and protein-ligand interaction study.

Cell-free expression-based screening is sometimes more suitable than cell-based assays for enzyme discovery. The advantage of cell-free systems for expression of toxic, poorly expressed, or insoluble proteins has already been well documented. Cell-free methods can advantageously replace cell-based ones when screening has to be performed on cell lysates prepared from harvested cells, for instance, when dealing with protein-ligand interactions particularly when the latter is hydrophobic.

[The prediction of immunogenicity of therapeutic proteins].

Immunogenicity of therapeutic proteins is a nightmare for industrials because induced antibodies can neutralize the therapeutic activity and provoke autoimmune symptoms. It was believed that sequence humanization would be sufficient to tackle these problems but multiple clinical examples now demonstrate that humanization does not suffice to abrogate immune responses. In order to predict immunogenicity of therapeutic proteins, different approaches have been developed, among which the most relevant ones are based on the evaluation of the response of naïve CD4 T lymphocytes specific for therapeutic proteins.

Quantitative analysis of the CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors.

Therapeutic antibodies are generally partially to fully humanized, yet they can show unwanted immunogenicity and lead to antibody response and adverse effects when administered to humans. As immunogenicity relies on a T-cell-dependent mechanism, we have evaluated in vitro the size of the preexisting CD4 T-cell repertoire specific to therapeutic antibodies in healthy donors. Specific CD4 T cells of individuals with different HLA-DR allotypes were amplified by in vitro stimulation and quantified.

Quantification of the preexisting CD4 T-cell repertoire specific for human erythropoietin reveals its immunogenicity potential.

Antibody-mediated pure red cell aplasia is a rare but serious event resulting from the induction of neutralizing erythropoietin (Epo)-specific antibodies provoked by treatment with recombinant Epo. Because of the crucial role of CD4 T cells in humoral response, we have quantified the number of Epo-specific CD4 T cells in the blood of normal donors by in vitro stimulation. An important repertoire of preexisting Epo-specific T cells was observed in almost half of the donors, comparable with that of non-self-proteins.

A quick in vitro pathway from prokaryotic genomic libraries to enzyme discovery.

Screening of prokaryotic genomes in order to identify enzymes with a desired catalytic activity can be performed in vivo in bacterial cells. We propose a strategy of in vitro expression screening of large prokaryotic genomic libraries based on Escherichia coli cell-free transcription-translation systems. Because cell-based expression may be limited by poor yield or protein misfolding, cell-free expression systems may be advantageous in permitting a more comprehensive screen under conditions optimized for the desired enzyme activity.

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants.

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined.

rdlA, a new gene encoding a rhodanese-like protein in Halanaerobium congolense and other thiosulfate-reducing anaerobes.

The recently described anaerobic moderately halophilic bacterium Halanaerobium congolense has been shown to reduce thiosulfate and sulfur-but not sulfate-into sulfide. When cultivated in the presence of thiosulfate as terminal electron acceptor, H. congolense possesses a highly active thiosulfate:cyanide sulfur-transferase activity (rhodanese-like enzyme).

Clostridium caminithermale sp. nov., a slightly halophilic and moderately thermophilic bacterium isolated from an Atlantic deep-sea hydrothermal chimney.

A strictly anaerobic, slightly halophilic and moderately thermophilic, sporulating rod designated strain DVird3T was isolated from deep-sea hydrothermal vent samples collected at a depth of approximately 800 m on the Atlantic Ocean Ridge. Strain DVird3T possessed a few laterally inserted flagella, had a DNA G + C content of 33.1 mol% and grew optimally at pH 6.