We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD 50 of freshly prepared whole venom of C. noxius (7.5 g/20 g of mouse) and C. suffusus (26.25 g/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.DNA supercoiling is essential for DNA metabolism. Supercoiling is introduced into DNA molecules by enzymes called DNA topoisomerases, which break, pass DNA strands through the break, and rejoin DNA (6,24,30). In Escherichia coli the level of supercoiling depends mainly on the activities of DNA topoisomerase I (TopI) and TopII (gyrase) and, to a lesser extent, on TopIV (6,30,32). TopI introduces DNA singlestrand breaks and relaxes DNA molecules. Gyrase, an ATPdependent enzyme composed of two GyrA and two GyrB subunits, makes double-strand breaks and introduces supercoils into DNA. TopIV is an ATP-dependent enzyme that makes double-strand breaks and contributes to DNA relaxation (24,30,32). The level of DNA supercoiling is regulated by a complex homeostatic control (19,28,32). Transcription of topA, which encodes TopI, increases when the level is high, whereas transcription of the gyr genes increases when the level is low (19,28). It is well established that changes in the level of DNA supercoiling influence the activity of many promoters (9,30) and that environmental variations alter this level (2,4,8,22,23). The role of DNA topoisomerases during the cellular response to these variations is not completely understood.E. coli stationary-phase cells in minimal or rich media, or after extreme nutrient downshifts, display a relaxation of DNA (2,8,26,28). In general, stationary-phase cells exhibit a number of morphological and physiological changes in order to survive starvation. These changes include resistance to several harmful conditions, condensation of the nucleoid, increased protein degradation, and a general decrease in transcription and translation (13). In E. coli many of these characteristics depend on the product of rpoS, S , a global regulator responsible for the induction of more than 50 genes (11,12). It has been shown that S -dependent promoter recognition is more efficient on relaxed DNA templates, suggesting that the DNA relaxation that occurs in stationary-phase cells plays a role in the transcription of S -dependent genes (14). As a first step to a better understanding of the regulation of DNA supercoiling during nutritional stress, the level of supercoiling and the expression of the gyr genes were determined along the growth curve and in stationary-phase cells after dilution into fresh medium. The role of S on the level of DNA supercoiling was also explored.Variations in the level of DNA supercoiling in exponentially growing cells, cells in stationary phase, and cells starved for several hours were determined by using plasmid pMS01,...

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.