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Results for “DNA Shuffling”

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At least 19 recordsLinked to original sources

PubMed1994

Rapid evolution of a protein in vitro by DNA shuffling.

DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. Computer simulations called genetic algorithms have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis and error-prone PCR are not combinatorial and thus are limited in searching sequence space. We have tested mutagenic DNA shuffling for molecular evolution in a beta-lactamase model system. Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 micrograms ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase.

Base Sequence↗

PubMed2002

Anticipatory evolution and DNA shuffling.

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.

DNA↗

PubMed2002

Evolution of a human immunodeficiency virus type 1 variant with enhanced replication in pig-tailed macaque cells by DNA shuffling.

DNA shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV) nef. Sequences spanning the gag-protease-reverse transcriptase (gag-pro-RT) region from several HIV-1 isolates were shuffled and cloned into a parental HIV-1 backbone containing SIV nef. Neither this full-length parent nor any of the unshuffled HIV-1 isolates replicated appreciably or sustainably in pt mPBMC. Upon selection of the shuffled viral libraries by serial passaging in pt mPBMC, a species emerged which replicated at substantially higher levels (50 to 100 ng/ml p24) than any of the HIV-1 parents and most importantly, could be continuously passaged in pt mPBMC. The parental HIV-1 isolates, when selected similarly, became extinct. Analyses of full-length improved proviral clones indicate that multiple recombination events in the shuffled region and adaptive changes in the rest of the genome contributed synergistically to the improved phenotype. This improved variant may prove useful in establishing a pig-tailed macaque model of HIV-1 infection.

Animals↗

PubMed1997

Applications of DNA shuffling to pharmaceuticals and vaccines.

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.

Amino Acid Sequence↗

PubMed1999

Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.

Base Sequence↗

PubMed2003

Computational and experimental analysis of DNA shuffling.

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless "junk" DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions.

Base Sequence↗

PubMed1997

Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening.

An efficient beta-fucosidase was evolved by DNA shuffling from the Escherichia coli lacZ beta-galactosidase. Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native beta-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for o-nitrophenyl 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 kcat/Km 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 shuffling 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.

Escherichia coli↗

PubMed2001

DNA shuffling and vaccines.

One challenge of biotechnology is to find ways to optimize enzymes, cytokines, vaccines or transgenes in new contexts that are typically not found in nature. The approach of DNA shuffling is a test-tube process that takes advantage of recombination to generate libraries of chimeric genes, which can then be screened to identify the encoded proteins improved in one or more functions. DNA shuffling of two or more genes that are structurally similar and homologous in function is particularly efficacious in generating large libraries of functionally novel molecules. Other directed evolution methods, such as those involving directed or random mutagenesis, have several limitations compared to the DNA shuffling recombination process. A wide variety of genes have been submitted to DNA shuffling, and significant improvements in various functional parameters have been obtained. Several examples of the application of DNA shuffling to vaccine development, therapeutics and gene therapy are discussed here.

Allergens↗

PubMed2002

Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase.

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.

DNA Shuffling↗

PubMed2002

Random DNA fragmentation with endonuclease V: application to DNA shuffling.

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.

DNA↗

PubMed1996

Improved green fluorescent protein by molecular evolution using DNA shuffling.

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.

Animals↗

PubMed2003

DNA shuffling method for generating estrogen receptor alpha and beta chimeras in yeast.

To facilitate our study of the molecular basis for the estrogen receptor (ER) subtype selectivity of novel ligands, we used DNA shuffling to construct chimeric ERs having ligand binding domains derived from both ER alpha and ER beta. The efficiency of chimera generation was low with traditional DNA shuffling protocols. Furthermore, ER ligand binding domain sequences lack convenient restriction sites for introducing chimeric ligand binding domain sequences into expression vectors. To overcome these problems, we developed a modified strategy whereby chimeric sequences were exclusively amplified from among the reassembled products from DNA shuffling using a special pair of PCR primers whose 3' ends specifically match the alpha and beta sequences, respectively, and whose 5' ends match sequences outside the ER beta ligand binding domain. When chimeric ligand binding domain DNA sequences, amplified with these primers, were co-transformed into a yeast strain with a linearized expression vector for ER beta, an active expression vector was produced by homologous recombination. Twenty-two different crossover sites were found; most occurred when there was a stretch of eight or more identical base pairs in both sequences, and many were concentrated in the regions important for studying ligand binding and transactivation. This method should prove to be useful for generating chimeric gene products from parent templates that share relatively low sequence identity.

Amino Acid Sequence↗

PubMed2002

Predicting out-of-sequence reassembly in DNA shuffling.

We present an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments. Out-of-sequence annealing events are undesirable since they typically encode non-functional proteins with missing or repetitive regions. The approach builds on the e Shuffle framework for the prediction of crossover formation using equilibrium thermodynamics and complete sequence information to model the reassembly process. An in silico case study of a set of subtilases reveals that, as expected, the presence of significant sequence identity between distant portions of the parental sequences gives rise to out-of-sequence annealing events that upon reassembly generate sequences with missing or repetitive DNA segments. The frequency of these events increases as the fragment length decreases. Interestingly, out-of-sequence annealing events are at a minimum near the annealing temperature of 55 degrees C used in the original DNA shuffling protocol. Neither parental sequence identity nor number of shuffled parents significantly alter the extent of out-of-sequence reassembly

Amino Acid Motifs↗

PubMed1997

Molecular evolution of an arsenate detoxification pathway by DNA shuffling.

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.

Adenosine Triphosphatases↗

PubMed2001

Predicting crossover generation in DNA shuffling.

We introduce a quantitative framework for assessing the generation of crossovers in DNA shuffling experiments. The approach uses free energy calculations and complete sequence information to model the annealing process. Statistics obtained for the annealing events then are combined with a reassembly algorithm to infer crossover allocation in the reassembled sequences. The fraction of reassembled sequences containing zero, one, two, or more crossovers and the probability that a given nucleotide position in a reassembled sequence is the site of a crossover event are estimated. Comparisons of the predictions against experimental data for five example systems demonstrate good agreement despite the fact that no adjustable parameters are used. An in silico case study of a set of 12 subtilases examines the effect of fragmentation length, annealing temperature, sequence identity and number of shuffled sequences on the number, type, and distribution of crossovers. A computational verification of crossover aggregation in regions of near-perfect sequence identity and the presence of synergistic reassembly in family DNA shuffling is obtained.

Algorithms↗

PubMed1997

Optimization of DNA shuffling for high fidelity recombination.

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.

Biological Evolution↗

PubMed1999

Evolution of a cytokine using DNA family shuffling.

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.

Amino Acid Sequence↗

PubMed2000

Breeding of retroviruses by DNA shuffling for improved stability and processing yields.

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.

Animals↗