The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5␤-or 5␣-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of threedimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5␤-or 5␣-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.coli clones with temperature-sensitive phenotype in the presence of chloramphenicol. The resistance to chloramphenicol used to select these clones results from expression control of the chloramphenicol acetyl transferase gene by the T7 promoter. In a second phase, and using the thermosensitive T7 RNAP variants as template, a new round of random mutagenesis was performed. Combined to an appropriate screening strategy, 11 mutations (second-site T7 RNAP revertants) that restore the initial resistance to chloramphenicol at 42°C were identified. Nine of these mutations increase the thermal resistance of the wild-type T7 RNA. They include the five mutations previously described using different approaches and four novel mutations. One improves T7 RNA catalytic activity and one has no positive effect on the natural enzyme but increases the activity of some combined mutants. Additive effects of mutations amount to an increase of as much as 10°C in T1/2 compared with the wild-type enzyme and up to a 2-fold activity enhancement.

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