The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3'-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with K d values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.E-selectin is a cell adhesion molecule which is induced on the surface of endothelial cells in response to inflammatory cytokines (1, 2). Binding of E-selectin to its ligand expressed on the surface of circulating neutrophils initiates rolling, an early step in the recruitment of these cells to a site of injury or inflammation (3, 4). The sequence of E-selectin (2,5,6) shows that it is a member of the mammalian C-type lectin family (7), and its three-dimensional structure shows strong similarity to mannose-binding protein (8). The carbohydrate structure sialyl Lewis x (sLe x ; 1 Neu5Ac␣2-3Gal␤1-4[Fuc␣1-3]GlcNAc) has been identified as a ligand which binds to the lectin domain at the N terminus of E-selectin (9 -12). However, natural ligands may also contain protein (13,14) or other carbohydrate structures. Inhibition of neutrophil adhesion to endothelium is an attractive approach to controlling inflammation-mediated diseases such as rheumatoid arthritis or psoriasis (15). Several potential therapeutics have been tested for their ability to inhibit the E-selectin-neutrophil adhesion event, including carbohydrate-based molecules (16, 17), antibodies (18), soluble E-selectin (19), and selectin-Ig chimeras (20). While these molecules have been useful to show the utility of selectin blockers for treating inflammation, each has significant drawbacks as a therapeutic, including short in vivo half-life, potential immunogenicity, high cost, and other possible side effects. A further limitation of these approaches is the lack of an efficient means to improve the pharmaceutical properties of these molecules.In the past few years, several methods for creating and screening vast libraries of recombinant peptides have been developed (21-24). These libraries have been used to discover novel peptide ligands for several proteins, including antibodies (21, 24, 25), receptors (26, 27), and lectins (28, 29), as well as novel enzyme substrates (30 -33). We report here the use of recombinant peptide dis...