共查询到20条相似文献,搜索用时 390 毫秒
1.
杨海杰 《国外医学:分子生物学分册》2001,23(2):121-125
体外DNAshuffling技术是一种快速、高效并且衫的分子定向进化技术,和以往的基因突变技术不同,它是一种高通量的突变筛选技术,可以对靶序列进行多次重组和选择,利用该技术建立的突变文库(嵌合文库)具有容量大、多样性好等优点,且更易于实现有益突变的积累。DNAshuffling技术已广泛应用于酶、药物蛋白及其它功能蛋白的改造,并取得了相当大的成功,显示了该技术在蛋白质改造和分子育种方面的潜在应用价值。 相似文献
2.
作为细胞结构与功能的中心参与者,蛋白质一直是生命科学研究的中心主题。分析蛋白质序列变异对其结构、功能的影响,是研究蛋白的重要手段之一。近年一种称为深度突变扫描(deep mutational scanning,DMS)的技术被广泛应用于蛋白研究领域,其通过高丰度DNA文库在蛋白特定区域平行引入成千上万种突变,经筛选后,利用高通量测序为每一种突变打分,从而揭示序列与功能之间的相关性。深度突变扫描以其高通量、快速简易、节省人工等特点,已经成为蛋白质功能研究以及蛋白工程改造的一种重要方法,目前已在蛋白进化、抗体改造、致病突变鉴定等蛋白研究的多个领域广泛应用。本综述简要概括了深度突变扫描技术的原理,重点介绍了其在哺乳动物细胞中的应用,同时分析了目前的技术瓶颈,旨在为相关研究提供参考。 相似文献
3.
4.
酶分子体外定向进化的研究进展 总被引:1,自引:0,他引:1
分子体外定向进化是改造酶分子的新策略,它主要通过体外模拟自然进化机制,利用基因随机突变、重组和定向筛选技术,使进化过程朝着人们需要的方向发展。简要介绍了酶分子体外定向进化的发展历史,详细介绍了突变文库构建和筛选方法的最新研究进展及应用情况。 相似文献
5.
6.
7.
在工业生物催化过程和生物细胞工厂构建方面,蛋白质定向进化被广泛地应用于酶的分子改造.蛋白质定向进化不仅可以针对某一目的蛋白进行改造,还可以改善代谢途径、优化代谢网络、获得期望表型细胞.为了获得更高效的突变效率,快捷、高通量的筛选方法,提高蛋白质定向进化的效果,研究者不断开发蛋白质体内、体外进化方法,取得了新的进展和应用.本文介绍了最近发展的蛋白质定向进化技术的原理、方法及特点,总结了突变文库的筛选方法和蛋白质定向进化的最新应用,最后讨论了蛋白质定向进化存在的挑战和未来发展方向. 相似文献
8.
9.
文库筛选与分子进化的核糖体展示新方法 总被引:3,自引:0,他引:3
利用适当的文库筛选技术快速、简便地从DNA文库、随机肽库、抗体库或其它蛋白文库中筛选生物活性物质是目前分子生物学研究的一个热点.核糖体展示是一种完全离体进行的功能蛋白筛选和进化鉴定的新技术,避免了传统的活体筛选技术的缺陷,使得文库容量增大、分子多样性加强.本文系统地评述了核糖体展示技术在制备ScFv单链抗体方面的应用,包括ScFv单链抗体模板的构建、体外转录与体外翻译、亲和筛选及筛选效率的测定以及分子多样性和体外进化研究,讨论了核糖体展示技术目前的发展动态、存在问题及发展趋势. 相似文献
10.
11.
Convergent evolution with combinatorial peptides 总被引:1,自引:0,他引:1
Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis. 相似文献
12.
Baolei Jia Gang-Won Cheong Shihong Zhang 《Extremophiles : life under extreme conditions》2013,17(2):193-203
Enzymes from many archaea colonizing extreme environments are of great interest because of their potential for various biotechnological processes and scientific value of evolution. Many enzymes from archaea have been reported to catalyze promiscuous reactions or moonlight in different functions. Here, we summarize known archaeal enzymes of both groups that include different kinds of proteins. Knowledge of their biochemical properties and three-dimensional structures has proved invaluable in understanding mechanism, application, and evolutionary implications of this manifestation. In addition, the review also summarizes the methods to unravel the extra function which almost was discovered serendipitously. The study of these amazing enzymes will provide clues to optimize protein engineering applications and how enzymes might have evolved on Earth. 相似文献
13.
The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. 相似文献
14.
Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones. 相似文献
15.
Zhao H 《Biotechnology and bioengineering》2007,98(2):313-317
Directed evolution has been successfully used to engineer proteins for basic and applied biological research. However, engineering of novel protein functions by directed evolution remains an overwhelming challenge. This challenge may come from the fact that multiple simultaneously or synergistic mutations are required for the creation of a novel protein function. Here we review the key developments in engineering of novel protein functions by using either directed evolution or a combined directed evolution and rational or computational design approach. Specific attention will be paid to a molecular evolution model for generation of novel proteins. The engineered novel proteins should not only broaden the range of applications of proteins but also provide new insights into protein structure-function relationship and protein evolution. 相似文献
16.
17.
Xiong AS Peng RH Zhuang J Davies J Zhang J Yao QH 《Critical reviews in biotechnology》2012,32(2):133-142
Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications. 相似文献
18.
链霉亲和素/生物素(Streptavidin/Biotin)体系作为目前已知的最高亲和力作用体系,已在生物学研究中获得广泛应用。本文针对Streptavidin/Biotin和Strep-Tactin/Strep-tag两个相关系统的演化,分别从链霉亲和素蛋白的结构改造、亲和肽标签优化等方面进行了较为详细的归纳。通过对链霉亲和素蛋白各种突变体的优缺点的比较,有助于实际应用中选择合适的Streptavidin突变体。本文通过对链霉亲和素蛋白质进化的综述,可帮助更准确地理解市场上各种链霉亲和素蛋白的功能和用途,并为深入研究链霉亲和素蛋白的进化提供参考。 相似文献
19.
Given the importance of protein-protein interactions for nearly all biological processes, the design of protein affinity reagents for use in research, diagnosis or therapy is an important endeavor. Engineered proteins would ideally have high specificities for their intended targets, but achieving interaction specificity by design can be challenging. There are two major approaches to protein design or redesign. Most commonly, proteins and peptides are engineered using experimental library screening and/or in vitro evolution. An alternative approach involves using protein structure and computational modeling to rationally choose sequences predicted to have desirable properties. Computational design has successfully produced novel proteins with enhanced stability, desired interactions and enzymatic function. Here we review the strengths and limitations of experimental library screening and computational structure-based design, giving examples where these methods have been applied to designing protein interaction specificity. We highlight recent studies that demonstrate strategies for combining computational modeling with library screening. The computational methods provide focused libraries predicted to be enriched in sequences with the properties of interest. Such integrated approaches represent a promising way to increase the efficiency of protein design and to engineer complex functionality such as interaction specificity. 相似文献
20.
Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression,
and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed
evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques
to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test
unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian
cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian
expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes
during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more
global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise
to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and
protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology. 相似文献