PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

An efficient ␤-fucosidase was evolved by DNA shuff ling from the Escherichia coli lacZ ␤-galactosidase. Seven rounds of DNA shuff ling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native ␤-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for onitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside. The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity. The evolved fucosidase has a 10-to 20-fold increased k cat ͞K m for the fucose substrates compared with the native enzyme. The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuff ling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.Proteins and enzymes with novel functions and properties can be obtained either by searching the largely unknown natural species or by improving upon currently known natural proteins or enzymes. The latter approach may be more suitable for creating properties for which natural evolutionary processes are unlikely to have been selected.One promising strategy to create such novel properties is by directed molecular evolution. Starting with known natural protein(s), multiple rounds of mutagenesis, functional screening, and amplification can be carried out. When the mutation rate, library size, and selection pressures are properly balanced, the desired phenotype of a protein generally increases with each round (1-8). The advantage of such a process is that it can be used to rapidly evolve any protein, without any knowledge of its structure.

Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pl258 encoded by genes arsR, arsB, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12-fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.

DNA shuffling of a family of over 20 human interferon-alpha (Hu-IFN-alpha) genes was used to derive variants with increased antiviral and antiproliferation activities in murine cells. A clone with 135,000-fold improved specific activity over Hu-IFN-alpha2a was obtained in the first cycle of shuffling. After a second cycle of selective shuffling, the most active clone was improved 285,000-fold relative to Hu-IFN-alpha2a and 185-fold relative to Hu-IFN-alpha1. Remarkably, the three most active clones were more active than the native murine IFN-alphas. These chimeras are derived from up to five parental genes but contained no random point mutations. These results demonstrate that diverse cytokine gene families can be used as starting material to rapidly evolve cytokines that are more active, or have superior selectivity profiles, than native cytokine genes.

We developed a chemically defined system to generate unlimited numbers of DCs from hESCs. Our results demonstrate that hESC-derived DCs generated from this process are immunogenic and have the potential to be used for DC immunotherapy.