Flexible synthesis and biological evaluation of novel 5-deoxyadenophorine analogues.

Adenophorine and its 5-deoxy analogue have been identified as natural iminosugars with efficient glycosidase inhibitory effects. The syntheses and biological evaluation of two new series of 5-deoxyadenophorine analogues in their racemic form are reported. The compounds 12e and 13d bearing a C11 and C7 alkyl chain, respectively, were found to be potent inhibitors of the beta-glucosidase from almond with Ki near to 60 microM. The compounds 13a,d which possess a 3,4-cis stereochemistry were efficient on glucosidases but also on the beta-galactosidase, what was not observed with the 3,4-trans series 12.

Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase.

The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.

Comparison of three microbial hosts for the expression of an active catalytic scFv.

Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.

Functional mimicry between anti-Tendamistat antibodies and alpha-amylase.

A proteinaceous inhibitor of alpha-amylase, Tendamistat, was evaluated as an immunogen to induce antibodies that mimic the enzyme activity and to investigate a new strategy to produce catalytic antibodies. Anti-Tendamistat polyclonal antibodies (pAbs) were shown to cross-react with acarbose, a strong carbohydrate inhibitor of alpha-amylases, and the alpha-amylase carbohydrate substrates, maltotetraose and maltoheptaose. Catalytic features of pAbs were characterized after denaturing natural serum alpha-amylase by treatment at pH 10 and in the presence of EDTA. A significant residual alpha-amylase activity was detected and associated with the IgG fraction, demonstrating that some antibodies behave as a functional mimic of natural amylase. Such antibodies, which behave as an "internal image" of the alpha-amylase, were used as immunogens to elicit anti-idiotype antibodies. It was demonstrated that the anti-idiotype antiserum contains a significant amount of antibodies that bind to porcine pancreatic amylase, which shows that they contain structural information from the original hapten, Tendamistat.