In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC 50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.affinity maturation ͉ human antibodies ͉ IL-13 ͉ protein evolution ͉ ribosome display

hsp83 was cloned from the filarial nematode Brugia pahangi. The mRNA was constitutively expressed at 37 8C in life cycle stages that live in the mammalian host (microfilariae and adult worms). Heat shock resulted in only a minimal increase in levels of transcription. A genomic copy of hsp83 was isolated and was shown to contain 11 introns while sequencing of the 5 0 upstream region revealed several heat shock elements. Using a chloramphenicol acetyltransferase (CAT) reporter gene construct the expression of hsp83 from B. pahangi (Bp-hsp83 ) was studied by transfection of COS-7 cells. Similar to the expression pattern in the parasite, CAT activity was detected at 37 8C and was not influenced by heat shock. When the free-living nematode Caenorhabditis elegans was transfected with the same construct, CAT activity was not observed at normal growth temperatures (21 8C) or under moderate heat shock conditions (28 8C). However exposure to more severe heat shock (35 8C) resulted in an increase in CAT activity. These results suggest that Bp-hsp83 has a temperature threshold $ 35 8C for expression.Keywords: Caenorhabditis elegans; CAT assay; heat shock elements; temperature threshold; transient transfection.Proteins of the heat shock protein (Hsp)90 family have been characterized extensively in yeast and higher eukaryotes, where they function both as molecular chaperones and as stress proteins [1,2]. In many cell types Hsp90 specifically associates with a range of regulatory proteins including steroid hormone receptors [3,4] and cyclin-dependant kinases [5]. Rutherford & Lindquist [6] proposed that the ability of Hsp90 to associate with signal transduction proteins, together with its capacity to act as a stress protein, provides a link between various developmental pathways and environmental change. Indeed they provided experimental evidence that Hsp90 is able to 'buffer' various mutant phenotypes in Drosophila and demonstrated that inhibition of Hsp90 allowed expression of otherwise silent mutations.The association of Hsp90 with components of the cell cycle provides a mechanism by which Hsp90 may be involved in developmental switching. In the free-living nematode Caenorhabditis elegans, hsp90 transcripts were shown to be 10 to15-fold enriched in the dauer larvae [7], while the transcription of other mRNAs tested was significantly reduced. The dauer larva is a developmentally arrested stage arising in response to adverse environmental conditions such as over-crowding or food shortages [8]. When worms were stimulated to emerge from dauer, hsp90 levels showed a marked decline within 2 h of recovery [7]. hsp90 in C. elegans has more recently been shown to be a member of the daf (dauer formation) gene family, daf-21. A gain of function mutant in daf-21 confers a dauerconstitutive phenotype suggesting that Hsp90 has a function in the dauer pathway, whereas the daf-21 null phenotype displays a growth arrest at the L2 to L3 stage [9].In this study, we report the cloning and characterization of the hsp90 homologue from the paras...