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1.
Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers 总被引:3,自引:1,他引:2
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Kawarasaki Y Griswold KE Stevenson JD Selzer T Benkovic SJ Iverson BL Georgiou G 《Nucleic acids research》2003,31(21):e126
SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity. 相似文献
2.
The method of incremental truncation for the creation of hybrid enzymes (ITCHY) allows the creation of comprehensive fusion libraries between 5 and 3 fragments of two genes in a manner that is independent of DNA sequence homology. A methodology is presented for the creation of ITCHY libraries called circularly permuted ITCHY (CP-ITCHY) that allows the creation of ITCHY libraries in a manner that does not require extensive time point sampling. In addition, CP-ITCHY requires only a single vector and productively biases the library towards those fusions that are approximately the same size as the original genes. In the model system of creating fusions between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, the CP-ITCHY libraries are shown to contain a diverse set of active fusions including those in regions of low-homology. In addition, a high percentage of active fusions were temperature-sensitive as they complemented an auxotrophic strain of Escherichia coli at 22 °C but not at 37 °C. 相似文献
3.
Ostermeier M 《Biotechnology and bioengineering》2003,82(5):564-577
Incremental truncation is a method for constructing libraries of every one base pair truncation of a segment of DNA. Incremental truncation libraries can be created using a time-dependent nuclease method or through the incorporation of alpha-phosphothioate dNTPs by PCR or by primer extension (THIO(pcr) truncation and THIO(extension) truncation, respectively). Libraries created by the fusion of two truncation libraries, known as ITCHY libraries, can be created using the above methods or by the incremental truncation-like method SHIPREC. Knowing and being able to tailor the distribution of truncations in incremental truncation, ITCHY and SHIPREC libraries would be beneficial for their use in protein engineering and other applications. However, the experimental determination of the distributions would require extensive, cost-prohibitive, DNA sequencing to obtain statistically relevant data. Instead, a theoretical prediction of the distributions was developed. Time-dependent incremental truncation libraries had the most uniform distribution of truncation lengths, but were biased against longer truncations. Essentially uniform distribution over the desired truncation range (from zero to N(max) base pairs) required that truncations be prepared up to at least 1.2-1.5 N(max). THIO(pcr) and THIO(extension) truncation libraries had a very nonuniform distribution of truncation lengths with a bias against longer truncations. Such nonuniformity could be significantly diminished by decreasing the incorporation rate of alphaS-dNTPs but at the expense of having a large fraction of the DNA truncated beyond the desired range or completely degraded. ITCHY libraries created using time-dependent truncation had the most uniform distribution of possible fusions and had the highest fraction of the library being parental-length fusions. However, the distribution of parental-length fusions was biased against fusions near the beginning/ends of genes unless the truncation libraries are prepared with a uniform distribution up to N(max). In contrast, SHIPREC libraries and THIO(pcr) ITCHY libraries, by the very nature of the nonuniform distributions of the truncated DNA, are ensured of having a uniform distribution of fusion points in parental-length fusions. This comes at the expense of having a smaller fraction of the library being parental-length fusions; however, this limitation can be overcome by performing size selection on the library. 相似文献
4.
Isabel Pardo Ana Isabel Vicente Diana M. Mate Miguel Alcalde Susana Camarero 《Biotechnology and bioengineering》2012,109(12):2978-2986
DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high‐redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α‐factor prepro‐leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high‐throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 2978–2986. © 2012 Wiley Periodicals, Inc. 相似文献
5.
In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships. 相似文献
6.
Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of alpha-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3'-->5' exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology. 相似文献
7.
Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution 总被引:4,自引:0,他引:4
Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space. 相似文献
8.
Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display 总被引:1,自引:0,他引:1
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Homologous recombination in yeast can be exploited to reliably generate libraries of >107 transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5′ homology to the cut vector and the second scFv PCR product lacked 3′ homology to the cut vector, or both PCR products had both 5′ and 3′ homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 104–105 transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast. 相似文献
9.
In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling 总被引:1,自引:0,他引:1
Xue-Fei Wang Qi-Zhang Li Ting-Wen Bao Wei-Ran Cong Wen-Xia Song Xuan-Wei Zhou 《Applied microbiology and biotechnology》2013,97(6):2455-2465
Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies. 相似文献
10.
Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification 总被引:2,自引:0,他引:2
A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling. 相似文献
11.
Rapid generation of incremental
truncation libraries for protein engineering using α-phosphothioate nucleotides
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Incremental truncation for the creation of hybrid enzymes (ITCHY) is a novel tool for the generation of combinatorial libraries of hybrid proteins independent of DNA sequence homology. We herein report a fundamentally different methodology for creating incremental truncation libraries using nucleotide triphosphate analogs. Central to the method is the polymerase catalyzed, low frequency, random incorporation of α-phosphothioate dNTPs into the region of DNA targeted for truncation. The resulting phosphothioate internucleotide linkages are resistant to 3′→5′ exonuclease hydrolysis, rendering the target DNA resistant to degradation in a subsequent exonuclease III treatment. From an experimental perspective the protocol reported here to create incremental truncation libraries is simpler and less time consuming than previous approaches by combining the two gene fragments in a single vector and eliminating additional purification steps. As proof of principle, an incremental truncation library of fusions between the N-terminal fragment of Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and the C-terminal fragment of human glycinamide ribonucleotide formyltransferase (hGART) was prepared and successfully tested for functional hybrids in an auxotrophic E.coli host strain. Multiple active hybrid enzymes were identified, including ones fused in regions of low sequence homology. 相似文献
12.
Directed evolution of proteins by exon shuffling 总被引:18,自引:0,他引:18
Evolution of eukaryotes is mediated by sexual recombination of parental genomes. Crossovers occur in random, but homologous, positions at a frequency that depends on DNA length. As exons occupy only 1% of the human genome and introns about 24%, by far most of the crossovers occur between exons, rather than inside. The natural process of creating new combinations of exons by intronic recombination is called exon shuffling. Our group is developing in vitro formats for exon shuffling and applying these to the directed evolution of proteins. Based on the splice frame junctions, nine classes of exons and three classes of introns can be distinguished. Splice frame diagrams of natural genes show how the splice frame rules govern exon shuffling. Here, we review various approaches to constructing libraries of exon-shuffled genes. For example, exon shuffling of human pharmaceutical proteins can generate libraries in which all of the sequences are fully human, without the point mutations that raise concerns about immunogenicity. 相似文献
13.
DNA改组的最新动态及应用前景 总被引:1,自引:0,他引:1
DNA改组(DNA shuffling)是目前最方便、有效的一种分子水平的体外定向进化技术,该技术同倾向错误PCR (Error-prone PCR) 相结合,通过对单基因或相关基因家族的靶序列进行多轮随机诱变、重组和高通量的筛选,可以有效富集正突变,去除负突变,提高突变文库的丰度,创造新基因和获得期望功能的蛋白质。DNA改组技术已在新药物等领域取得了广泛的应用,极大地推动了现代生物科学和生物技术的发展。该技术同计算机强大的数据分析系统相结合,将会为后基因组学的发展提供强有力的技术平台。 相似文献
14.
Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently 总被引:7,自引:0,他引:7
Ness JE Kim S Gottman A Pak R Krebber A Borchert TV Govindarajan S Mundorff EC Minshull J 《Nature biotechnology》2002,20(12):1251-1255
We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods. 相似文献
15.
Somatic cell hybrids exhibiting co-expression of the globin genes of two species were generated by fusion of mouse erythroleukemia cells with Chinese hamster or human marrow erythroid cells. In contrast, extinction of the mouse globin genes occurred in hybrids formed between the erythroleukemia cells and human fibroblasts. Direct detection of the human globin genes in human X mouse fibroblast hybrids was achieved by annealing of DNA from these cells to human globin complementary DNA. This method was developed to permit the chromosomal assignment of the human globin genes. 相似文献
16.
17.
Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto 总被引:1,自引:0,他引:1
Yongjun C Wei B Shujun J Meizhi W Yan J Yan Y Zhongliang Z Goulin Z 《FEMS microbiology letters》2011,325(2):155-161
Nattokinase (subtilisin NAT, NK) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from Bacillus natto. In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B. natto AS 1.107, Bacillus amyloliquefaciens CICC 20164 and Bacillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate-based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild-type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate-binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy. 相似文献
18.
The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity. 相似文献
19.
Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system 总被引:14,自引:0,他引:14
T E Petersen M R Martzen A Ichinose E W Davie 《The Journal of biological chemistry》1990,265(11):6104-6111
The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed. 相似文献
20.
Abécassis V Jaffrelo L Rickman D Aggerbeck L Herbert C Truan G Pompon D 《BioTechniques》2003,34(6):1272-1279
Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution. 相似文献