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PubMed2006

Exploiting directed evolution for the discovery of biologicals.

Protein-based drugs offer unique advantages over small-molecule drugs in terms of both discovery and therapeutic use. The advent of recombinant DNA technology enabled the production of recombinant proteins and the generation of partially or fully human monoclonal antibodies, and continued developments in molecular biology have provided powerful approaches to generate improved proteins with more drug-like features. In this review, the benefits of using an evolutionary approach to biologicals drug discovery are discussed, with emphasis on the use of in vitro evolution technologies, such as ribosome display.

Animals↗
PubMed2005

Directed evolution of (betaalpha)(8)-barrel enzymes.

Natural molecular evolution supplies us with manifold examples of protein engineering. The imitation of these natural processes in the design of new enzymes has led to surprising and insightful results. Well-suited for design by evolutionary methods are enzymes with the common and versatile (betaalpha)(8)-barrel fold. Studies of enzyme stability, folding and design as well as the evolution of (betaalpha)(8)-barrel enzymes are discussed.

Animals↗
PubMed2004

Directed evolution of nucleic acid enzymes.

Just as Darwinian evolution in nature has led to the development of many sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful approach for obtaining similar results in the laboratory. This review focuses on the development of nucleic acid enzymes starting from a population of random-sequence RNA or DNA molecules. In order to illustrate the principles and practice of in vitro evolution, two especially well-studied categories of catalytic nucleic acid are considered: RNA enzymes that catalyze the template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of RNA. The former reaction, which involves attack of a 2'- or 3'-hydroxyl on the alpha-phosphate of a 5'-triphosphate, is more difficult. It requires a comparatively larger catalytic motif, containing more nucleotides than can be sampled exhaustively within a starting population of random-sequence RNAs. The latter reaction involves deprotonation of the 2'-hydroxyl adjacent to the cleavage site, resulting in cleaved products that bear a 2',3'-cyclic phosphate and 5'-hydroxyl. The difficulty of this reaction, and therefore the complexity of the corresponding DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal cation or small molecule, is present in the reaction mixture.

DNA, Catalytic↗
PubMed2005

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus.

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.

Base Sequence↗
PubMed2006

Directed evolution to enhance secretion efficiency and thermostability of chitosanase from Mitsuaria chitosanitabida 3001.

Chitosanase (ChoA) from Mitsuaria chitosanitabida 3001 was successfully evolved with secretion efficiency and thermal stability. The inactive ChoA mutant (G151D) gene was used to mutate by an error-prone PCR technique and mutant genes that restored chitosanase activity were isolated. Two desirable mutants, designated M5S and M7T, were isolated. Two amino acids, Leu74 and Val75, in the signal peptide of ChoA were changed to Gln and Ile respectively in the M7T mutant, in addition to the G151D mutation. The L74Q/V75I double ChoA mutant was 1.5-fold higher in specific activity than wild-type ChoA due to efficient secretion of ChoA. One amino acid Asn222 was changed to Ser in the M5S mutant in addition to the G151D mutation. The N222S single ChoA mutant was 1.2-fold higher in specific activity and showed a 17% increase in thermal stability at 50 degrees C as compared with wild-type ChoA. This is the first study to achieve an evolutional increase in enzyme capability among chitosanses.

Burkholderia↗
PubMed2001

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Base Sequence↗
PubMed2004

A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge.

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide-mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in "sequence spaces" ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of -1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.

Amino Acid Sequence↗