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PubMed2000

Modeling DNA mutation and recombination for directed evolution experiments.

Directed evolution experiments rely on the cyclical application of mutagenesis, screening and amplification in a test tube. They have led to the creation of novel proteins for a wide range of applications. However, directed evolution currently requires an uncertain, typically large, number of labor intensive and expensive experimental cycles before proteins with improved function are identified. This paper introduces predictive models for quantifying the outcome of the experiments aiding in the setup of directed evolution for maximizing the chances of obtaining DNA sequences encoding enzymes with improved activities. Two methods of DNA manipulation are analysed: error-prone PCR and DNA recombination. Error-prone PCR is a DNA replication process that intentionally introduces copying errors by imposing mutagenic reaction conditions. The proposed model calculates the probability of producing a specific nucleotide sequence after a number of PCR cycles. DNA recombination methods rely on the mixing and concatenation of genetic material from a number of parent sequences. This paper focuses on modeling a specific DNA recombination protocol, DNA shuffling. Three aspects of the DNA shuffling procedure are modeled: the fragment size distribution after random fragmentation by DNase I, the assembly of DNA fragments, and the probability of assembling specific sequences or combinations of mutations. Results obtained with the proposed models compare favorably with experimental data.

Animals↗
PubMed2002

Predicting evolution by in vitro evolution requires determining evolutionary pathways.

In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 beta-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

Alleles↗
PubMed1999

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.

Amino Acid Sequence↗
PubMed2005

Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains.

DNA shuffling and other in vitro recombination strategies have proven highly effective at generating complex libraries for mutagenesis studies. While most recombination techniques employ DNA polymerases in part of a multi-step process, few seek to exploit the natural recombinogenic tendencies and exponential amplification rates of PCR. Here, we characterize a simple but effective method for using standard PCR to promote high recombination frequencies among compact heterologous domains by locating the domains near one end of the template. In a typical amplification reaction, Pfu polymerase generated chimeric crossover events in 13% of the population when markers were separated by only 70 nt. The fraction of recombinant sequences reached 42% after six consecutive rounds of PCR, a value close to the 50% expected from a fully shuffled population. When homology within the recombinant region was reduced to 82%, the recombination frequency dropped by nearly half for a single amplification reaction and crossover events were clustered toward one end of the domain. Surprisingly, recombination frequencies for template populations with high and low sequence homologies converged after just four rounds of PCR, suggesting that the exponential accumulation of chimeric molecules in the PCR mixture serves to promote recombination within heterologous domains.

DNA Shuffling↗
PubMed2002

DNA family shuffling of hyperthermostable beta-glycosidases.

The structural compatibility of two hyperthermostable family 1 glycoside hydrolases, Pyrococcus furiosus CelB and Sulfolobus solfataricus LacS, as well as their kinetic potential were studied by construction of a library of 2048 hybrid beta-glycosidases using DNA family shuffling. The hybrids were tested for their thermostability, ability to hydrolyse lactose and sensitivity towards inhibition by glucose. Three screening rounds at 70 degrees C led to the isolation of three high-performance hybrid enzymes (hybrid 11, 18 and 20) that had 1.5-3.5-fold and 3.5-8.6-fold increased lactose hydrolysis rates compared with parental CelB and LacS respectively. The three variants were the result of a single crossover event, which gave rise to hybrids with a LacS N-terminus and a main CelB sequence. Constructed three-dimensional models of the hybrid enzymes revealed that the catalytic (betaalpha)(8)-barrel was composed of both LacS and CelB elements. In addition, an extra intersubunit hydrogen bond in hybrids 18 and 20 might explain their superior stability over hybrid 11. This study demonstrates that extremely thermostable enzymes with limited homology and different mechanisms of stabilization can be efficiently shuffled to form stable hybrids with improved catalytic features.

Amino Acid Sequence↗
PubMed2005

Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.

Animals↗
PubMed2001

Creating multiple-crossover DNA libraries independent of sequence identity.

We have developed, experimentally implemented, and modeled in silico a methodology named SCRATCHY that enables the combinatorial engineering of target proteins, independent of sequence identity. The approach combines two methods for recombining genes: incremental truncation for the creation of hybrid enzymes and DNA shuffling. First, incremental truncation for the creation of hybrid enzymes is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. SCRATCHY libraries were created from the glycinamide-ribonucleotide formyltransferase (GART) genes from Escherichia coli (purN) and human (hGART). The developed modeling framework eSCRATCHY provides insight into the effect of sequence identity and fragmentation length on crossover statistics and draws contrast with DNA shuffling. Sequence analysis of the naive shuffled library identified members with up to three crossovers, and modeling predictions are in good agreement with the experimental findings. Subsequent in vivo selection in an auxotrophic E. coli host yielded functional hybrid enzymes containing multiple crossovers.

Algorithms↗
PubMed2006

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Allergens↗
PubMed1996

Base eversion and shuffling by DNA methyltransferases.

The structures of two DNA cytosine methyltransferases reveal two novel methods of gaining access to the substrate cytosine residue, both of which involve eversion of the cytosine in a process that may require DNA bending. In one instance there is also widespread base shuffling and distortion of the DNA.

DNA Modification Methylases↗
PubMed2000

An effective family shuffling method using single-stranded DNA.

Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fragments into parental gene sequences, because such a process prevents the formation of chimeric sequences. In order to improve the efficiency of hybrid formation in family shuffling, single-stranded DNAs (ssDNAs) were used as templates. The ssDNAs of two catechol 2,3-dioxygenase genes, nahH and xylE, were prepared, the xylE strand being complementary to the nahH strand. When these ssDNAs were digested by DNase I and reassembled, chimeric genes were obtained at a rate of 14%, which was much higher than the rate of less than 1% obtained by shuffling with double-stranded DNAs. Chimeric catechol 2,3-dioxygenases that were more thermally stable than the parental enzymes, XylE and NahH, were obtained by this ssDNA-based DNA shuffling.

Catechol 2,3-Dioxygenase↗
PubMed2013

Broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the GP4 or M genes.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.

Animals↗
PubMed2001

Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains.

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains. We first describe the "rational" design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies. The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.

Amino Acid Sequence↗
PubMed1998

Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system.

Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr1a (also known as mdr3) and mdr1b (also known as mdr1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [125I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Directed evolution and characterization of a novel D-pantonohydrolase from Fusarium moniliforme.

D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of D-pantoic acid. The Fusarium moniliforme D-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type D-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved D-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved D-pantonohydrolase were also studied and compared.

4-Butyrolactone↗
PubMed2005

Functional promoter analysis using an approach based on an in vitro evolution strategy.

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter function. The "selective-shotgun" approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning" deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the prospect of identifying interacting promoter regions.

Cloning, Molecular↗
PubMed2005

[Improving the affinity of an anti-TNF-alpha scFv by random mutation].

AIM: To improve the affinity of an anti-TNF-alpha scFv. METHODS: A mutant phage antibody library derived from an anti-TNF-alpha scFv gene was generated by error-prone PCR. The mutated genes were then subjected to DNA shuffling. Mutants with improved affinity were selected by bio-panning. Affinity improvement of the selected mutants was verified by dot blot ELISA and thiocyanate elusion ELISA. RESULTS: One mutant was obtained with relative affinity index (1.37 mol/L) higher than that of the parent scFv (0.48 mol/L). CONCLUSION: Error-prone PCR plus DNA shuffling is effective in improving the affinity of antibodies.

Amino Acid Sequence↗
PubMed2006

High efficiency and throughput system in directed evolution in vitro of reporter gene.

In vitro directed evolution, especially with DNA shuffling, is a powerful means in biological studies of protein structure and function, and consequently for industrial applications. Escherichia coli beta-glucuronidase (gusA) gene, a versatile and efficient reporter gene, was the model for studying in vitro directed evolution because of its stability, easy analysis of the enzyme properties and conveniently visible phenotype. We developed a high efficiency, throughput system for in vitro directed evolution using gusA reporter gene as the model. The system consisted mainly of three aspects: a prokaryotic expression vector pYPX251, an easy method for obtaining the mutated gene from DNA shuffling and a suitable selected strategy. The vector pYPX251 carried the moderately strong aacC1 gene promoter and T1T2 transcription terminator that allowed expression in E. coli. Over 10,000 individuals could be selected individually in a 9 cm Petri dish after colonies were absorbed on a nitrocellulose filter. A library, which contained 100,000 individuals was screened by incubating ten filter papers with X-Glu. The polymerase chain reaction products of the gusA gene, the fragments of 50-100 bp, with high mutation rates were purified using a dialysis bag from 10% PAGE after electrophoresis. The possibility of obtaining desirable mutations was increased dramatically as the size of the library expanded. A GUS variant, named GUS-TR, was obtained through this system, which is significantly more resistant to high temperature than the wild type enzyme. GUS-TR maintained its high activity even when the nitrocellulose filter containing the variant colony was heated at 100 degrees C for 30 min.

Biotechnology↗
PubMed2004

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol.

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.

Binding Sites↗