Shuffled: a software suite that assists the analysis of recombinant products resulting from DNA shuffling.
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DNA Shuffling↗
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A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display.
Antibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.
Amino Acid Sequence↗
DNA shuffling method for generating highly recombined genes and evolved enzymes.
We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.
Alleles↗
Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling.
The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.
Base Sequence↗
Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling.
A library of variant enzymes was created by combined shuffling of the DNA encoding the human Mu class glutathione transferases GST M1-1 and GST M2-2. The parental GSTs are 84 % sequence identical at the protein level, but their specific activities with the substrates aminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold. Aminochrome is of particular interest as an oxidation product of dopamine and of possible significance in the etiology of Parkinson's disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds. GST M2-2 has at least two orders of magnitude higher catalytic activity with both of the substrates than any of the other known GSTs, including GST M1-1. The DNA library of variant Mu class GST sequences contained "mosaic" structures composed of alternating segments of both parental sequences. All clones contained the 5'-end of a GST M1-1 clone optimized for high-level expression in Escherichia coli. The remainder of the sequences derived from segments of GST M2-2 and GST M1-1 DNA. All of the clones analyzed contained between two and seven distinct DNA segments. In addition, each clone contained an average of approximately one point mutation. None of the library clones analyzed was identical with either of the two parental structures. Variant GST sequences were expressed in E. coli, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determined in bacterial lysates. Such screening of more than 70 clones demonstrated a continuous range of activities covering at least two orders of magnitude for each of the substrates. For a given clone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB. This functional correlation suggests a common structural basis for the enzymatic mechanisms for conjugation of aminochrome and denitrosation of cyanoDMNG. From an evolutionary perspective, the results show that recombination of segments from homologous proteins gives rise to a large proportion of functionally competent proteins with a range of activities. The data support the proposal that natural evolution of protein functions may involve recombination of DNA segments followed by selection for advantageous functional properties of the resulting proteins. Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution.
Amino Acid Sequence↗
[Directed evolution of D-lactonohydrolase by error prone PCR and DNA shuffling].
D-lactonohydrolase is useful in the procedure of resolution of racemic pantolactone to produce D-pantolactone, but the activity and stability under low pH of the wild type enzyme is not satisfactory enough to be applied to industrial production. The expected properties of wild type enzyme were enhanced by directed evolution. According to the formation of products and pH indicators, a screening system was designed. After three sequential error prone PCR and one round DNA shuffling followed by screening, Mut E-861, the best mutant with improved activity and stability under low pH situation was obtained. Gene analysis of the Mut E-861 mutant indicated that the mutant enzyme had A352C, G721A mutations and a silent mutation of position 1038. Moreover, the activity and stability of Mut E-861 were determined. The results showed that the activity of this mutant was 5.5-fold higher than that of wild type, and the stability under low pH was improved at no expense of D-lactonohydrolase activity. After incubated at pH 6.0 and pH 5.0 the activity of D-lactonohydrolase could be retained 75% to 50%, however, compared with 40% to 20% for wild type.
Carboxylic Ester Hydrolases↗
Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis.
The avermectin analog doramectin (CHC-B1), sold commercially as Dectomax, is biosynthesized by Streptomyces avermitilis. aveC, a gene encoding an unknown mechanistic function, plays an essential role in the production of doramectin (avermectin CHC-B1), modulating the production ratio of CHC-B1 to other avermectins, most notably the undesirable analog CHC-B2. To improve the production ratio for doramectin, the aveC gene was subjected to iterative rounds of semi-synthetic DNA shuffling. Libraries of shuffled aveC gene variants were transformed into S. avermitilis, screened using a miniaturized 96-well growth and production format, and analyzed by high throughput mass spectrometry to determine CHC-B2:CHC-B1 ratios. Several improved aveC variants were identified; the best shuffled gene encoded 10 amino acid mutations, and conferred a final CHC-B2:CHC-B1 ratio of 0.07:1, a 23-fold improvement over the starting gene (aveC wild type). Chromosomal insertion of an improved aveC shuffled gene into a high titer S. avermitilis strain yielded an improved doramectin production strain. This strain is under development to be used commercially, and is expected to provide considerable cost savings in large-scale manufacture, as well as significantly reducing by-product levels of CHC-B2 requiring disposal.
DNA Shuffling↗
DNA shuffling as a tool for protein crystallization.
The success of structural studies performed on an individual target in small scale or on many targets in the system-wide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression, solubility, or crystallization. The method is scaleable and can be applied in situations where a single protein is being studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to structural studies of soluble proteins, membrane proteins, and macromolecular complexes.
Acetyltransferases↗
Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling.
To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.
Amino Acid Sequence↗
Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling.
DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-(3',4'-dichlorophenyl)glutarate (DDG). Three homologous lipases from Candida antarctica ATCC 32657, Hyphozyma sp. CBS 648.91 and Crytococcus tsukubaensis ATCC 24555 were cloned and shuffled to generate a diverse gene library. A high-throughput screening assay was developed and used successfully to identify chimeric lipase B proteins having a 20-fold higher activity toward DDG than lipase B from C.antarctica ATCC 32657 and a 13-fold higher activity than the most active parent derived from C.tsukubaensis ATCC 24555. In addition, the stability characteristics of several highly active chimeric proteins were also improved as a result of family shuffling. For example, the half-life at 45 degrees C and melting point (T(m)) of one chimera exceeded those of lipase B from C.antarctica ATCC 32657 by 11-fold and 6.4 degrees C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91.
Amino Acid Sequence↗
[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].
AIM: To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method. METHODS: PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin. RESULTS: After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained. CONCLUSION: Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.
Animals↗
Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus.
Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.
DNA, Viral↗
Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling.
Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.
Animals↗
Identification of an effector specificity subregion within the aromatic-responsive regulators DmpR and XylR by DNA shuffling.
The Pseudomonas derived sigma(54)-dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenol-binding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.
Amino Acid Sequence↗
Stabilities of chimeras of hyperthermophilic and mesophilic glycerol kinases constructed by DNA shuffling.
Glycerol kinases from Thermococcus kodakaraensis KOD1 (Tk-GK) and Escherichia coli (Ec-GK) greatly differ in thermostability. The temperature (T(1/2)) at which the enzymes lose half of their activity upon incubation for 20 min is 50-55 degrees C for Ec-GK and approximately 95 degrees C for Tk-GK. To examine whether the amino acid substitutions that make Tk-GK more stable than Ec-GK are localized in a limited region, the chimeras of two parental genes encoding Tk-GK and Ec-GK were constructed by DNA shuffling. E. coli cells were transformed with a plasmid library harboring these chimeras and screened for those tht produce chimeric enzymes which are more stable than Ec-GK. Four chimeric enzymes were isolated and purified, and their biochemical properties characterized. Replacement of 83 or 93 residues in the C-terminus of Ec-GK with the corresponding ones of Tk-GK increased the T(1/2) value of Ec-GK by 25-30 degrees C. In contrast, replacement of 85 residues in the N-terminus of Ec-GK with the corresponding ones of Tk-GK reduced the T(1/2) value by 5-10 degrees C. In addition, replacement of 10 residues in the C-terminus of Tk-GK with the corresponding ones of Ec-GK reduced the T(1/2) value ot Tk-GK by approximately 15 degrees C. Measurement of the far-UV CD spectra indicates that the three-dimensional structures of the chimeric enzymes, as well as those of the parent enzymes, are similar to one another. These results suggest that the amino acid substitutions responsible for the high stability of Tk-GK are largely localized in the C-terminal region.
Journal Article↗
Heterologous overexpression of a mutant termite cellulase gene in Escherichia coli by DNA shuffling of four orthologous parental cDNAs.
Among cellulase genes, those of animals are known for their difficulty in overexpression. We constructed a chimeric library by family shuffling of endo-beta-1,4-glucanase genes from four different termite species (Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus, and Coptotermes acinaciformis) sharing 78.5-96% homology in amino acid sequence. The constructed library was screened by Congo red plate assay combined with 96-well micro-enzyme assay, and clones showing enhanced CMCase activities were obtained. The mutated genes were overexpressed in Escherichia coli intracellularly as an active form. The endo-beta-1,4-glucanase (CMCase) activity in soluble fractions of E. coli harboring the mutant genes was 20-30 fold higher than that of wild-type genes. The mutant enzyme showed high activity against CMC and properties similar to those of the native enzymes.
Amino Acid Sequence↗
How DNA shuffling works.
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DNA, Recombinant↗