DNA改组技术在酶工程中的应用

DNA改组技术是一种应用最广泛且简单有效的分子定向进化方法.它是一种高通量的随机突变筛选技术,可以对目的基因进行多次重组和选择,得到性质优良的突变文库.介绍了DNA改组技术的原理及其在酶工程中的应用.     

蛋白质定向进化的研究进展及其应用前景

定向进化是改造蛋白质分子的有效新策略.它不需要了解蛋白质的空间结构,主要通过在实验室里模拟自然进化过程,采用错误倾向PCR等方法对编码蛋白质的基因进行随机突变,经DNA改组、交错延伸等技术进行体外重组,设计高通量筛选方法来选出需要的突变体.本综述了定向进化技术的发展及应用.     

蛋白质定向进化技术的研究进展

蛋白质定向进化技术是蛋白质分子改造的一个重要策略.重点介绍了易错PCR、DNA改组等对编码蛋白质的基因进行随机突变和重组的技术,以及构建突变体库和高通量筛选的方法,并探讨了定向进化技术在蛋白质工程中的应用及前景.     

蛋白质突变体基因库构建方法的研究进展

体外定向进化是蛋白质工程中一个非常有效的设计策略。最近几年,在过去常用的寡核苷酸介导的随机突变、易错PCR和DNA改组等方法的基础上又出现了一些新的定向进化方法。本文对这些方法及其特点加以总结,为解决特定问题选取何种方法提供一定依据。最近研究表明：定向进化和理性设计相结合、定向进化和以结构为基础的计算设计方法相结合正成为蛋白质工程中两个新的发展方向。     

透明颤菌血红蛋白的DNA改组研究

为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先通过易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造。将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库。以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb'042506。该蛋白使宿主的菌体湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序和比对,该基因与原基因相比发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱实验显示该蛋白具有更强的特征吸收。     

分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

[目的]红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源.为了提高该酶的性能,有必要对其进行定向进化.[方法]M-TreS基因(M-treS)大小为2 889bp.该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling.分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法.该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling; (3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库.[结果]结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株.序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变.[结论]分段DNA shuffling是进化大分子蛋白质的有效方法.     

用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants）,这些突变体中包含大量的限制性内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

DNA改组技术在水蛭素实验进化中的应用

蛋白质的改造是生物工程的重大研究课题.由于结构和功能预测的不精确性,而使按照三维结构信息进行定位诱变往往达不到预期的目的.近年来,另一条改造蛋白质的途径有较大的发展,即在实验室条件下模拟生物分子的自然进化,通过变异和靶功能的选择来获得改进性能的蛋白质[1],此过程称为生物分子实验定向进化.DNA改组(DNAshuffling)是一种改造基因和蛋白质的有效实验进化技术[2].它是在体外进行基因随机片段的重组,从而增加基因的多样性,促使有利变异与不利变异分离,通过选择使有利变异得到优化组合[3].DNA改组包含3个步骤:基因的随机片段化,自身引发PCR和重组合PCR.经过DNA改组的突变体库有可能选择到性能更优的突变体.为进行亲和淘选,需将突变体展示在噬菌体的表面[4].     

通过原位易错PCR一步构建基因突变文库

主要介绍一种通过原位易错PCR构建随机突变文库的新技术。本实验室最近发表的一项国际专利中,利用来源于海栖热孢菌的极耐热性DNA连接酶,在传统PCR循环中加入一个连接步骤,即变性—退火—延伸—连接的四步循环法PCR,从而实现环状质粒的PCR指数扩增(PPCP)。原位易错PCR中所用引物为一段线性双链DNA,它含有与模板质粒不同的筛选标记,产物转化宿主菌后,模板质粒在筛选平板上被直接剔除。筛选到的阳性突变子可用作模板直接进入突变文库的再次构建,通过筛选获得二级或多级累加的正突变。利用这种方法构建了一个木聚糖酶基因和一个纤维素酶基因的随机突变文库,并筛选出具有正向突变的蛋白,证明以PPCP为基础的原位易错PCR技术,为基因定向进化提供了一种快速有效的随机突变文库构建的新方法。     

一步法定点突变技术快速构建bsh基因突变启动子

目的：建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法：以构建单核细胞增生李斯特菌（Listeria monocytogenes）中编码胆碱水解酶（bile salt hydrolase,BSH）的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果：通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论：该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。     

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.     

DNA shuffling: Modifying the hand that nature dealt

Summary DNA shuffling is a technique being utilized for in vitro recombination of a single gene or pools of homologous genes. The genes are fragmented into randomly sized pieces, and polymerase chain reaction (PCR) reassembly of full-length genes from the fragments, via self-priming, yields recombination due to PCR template switching. After these PCR products are screened and the interesting products sequenced, improved clones are reshuffled to recombine useful mutations in additive or synergistic ways, in effect mimicking the process of natural sexual recombination. Proteins can be ‘bred’ with the appropriate individual properties and then their ‘progeny’ screened for the desired combination of traits. DNA shuffling is a powerful tool enabling rapid and directed evolution of new genes, operons and whole viral genomes.     

DNA shuffling技术及其在基因工程疫苗中的应用

DNA shuffling技术是一项全新的体外人工进化模式,它通过基因在分子水平上的重组,再定向筛选具有预期性状的突变体,获得同时具有多个亲本基因的特征的突变基因。该文介绍DNA shuffling技术的基本原理,并列举了由该技术发展而来的新技术及其在基因工程疫苗领域的应用,展望了DNAshuffling技术的发展方向。     

Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.

Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.     

A comparison of directed evolution approaches using the beta-glucuronidase model system

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.     

DNA改组(DNA shuffling)及其研究进展

自1994年首次提出至今,DNA改组已经成为定向进化最为有效的方法之一.无论DNA、蛋白质还是生物体的进化,DNA改组都有十分突出的表现.通过对十余年来DNA改组的发展作以简要综述,为日后相关研究的开展提供理论基础.     

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85 779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.     

蛋白质定向进化的研究进展

定向进化是改造蛋白质分子的一种有效的新策略。主要是在实验室里模拟自然进化过程,通过由易错PCR、致突变菌株诱变等方法对编码蛋白质的基因进行随机诱变,由DNA改组、随机引导重组和交错延伸等方法进行突变基因体外重组,设计高通量筛选方法来选出需要的突变株。它不仅可快速产生工业上有用的新酶,而且对研究蛋白质的结构与功能的关系具有非常重要的意义。     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.