多形汉逊酵母研究进展

作为研究甲醇代谢、过氧化物酶体稳态和硝酸盐吸收的模式生物,多形汉逊酵母近年来在基础研究领域日益受到重视。在工程应用领域,利用多形汉逊酵母表达真核外源基因有特殊的优势。譬如容易得到高拷贝,在含油酸的培养条件下能够表达膜蛋白等。已有多种外源蛋白在多形汉逊酵母系统中得到表达。本文综述了多形汉逊酵母的基本生物学性质、基础研究领域概况及其在外源基因表达方面的特点和进展。     

毕赤酵母表达系统

选择合适的表达系统是蛋白质体外成功表达的关键。毕赤酵母表达系统是一种新的、极具潜力的真核表达系统,其在蛋白质表达方面具有其它系统不可比拟的优点,正在得到越来越广泛的应用。较详细地综迷了毕赤酵母表达系统的特点、组成及影响其表达的因素等。     

Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins

We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.     

Study of animal viruses in yeast

Yeast is often considered to be a model eukaryotic organism, in a manner analogous to E. coli as a model prokaryotic organism. Yeast has been extensively characterized and the genomes completely sequenced. Despite the small genome size, yeast displays most of features of higher eukaryotes. The facts that most of cellular machinery is conserved among different eukaryotes and that the powerful technologies of genetics and molecular biology are available have made yeast model eukaryotic cells in biological and biomedical sciences including virology. Cumulative data indicate that yeast can be a host for animal viruses. I briefly describe yeast gene expression and review viral replication in yeast. Great discovery include complete replication of animal viruses and production of virus-like particle vaccines in yeast. Current studies on yeast focus on identification of host factors and machinery used for viral replication. The studies are based on traditional yeast genetics and genome-wide identification using a complete set of yeast deletion strains.     

InteBac: An integrated bacterial and baculovirus expression vector suite

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time‐consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N‐ and C‐terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost‐intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.     

重组蛋白表达系统的研究进展

利用基因重组表达外源蛋白在现代生物学技术的发展与应用中起着重要的作用。根据外源基因表达宿主不同,可以将表达系统大致分为两类:原核和真核表达系统。该文比较了常见的几种表达系统的优缺点,并探讨了在选择适合的表达系统时所需要考虑的因素如目标蛋白的产量、生物学活性、用途及其物理化学性质以及表达系统本身的成本、便利性及其安全性等,以便于选择适合的表达系统,优化提高重组蛋白产量等,进而更好地服务于科学研究。     

Production of membrane proteins using cell–free expression systems

Different overexpression systems are widely used in the laboratory to produce proteins in a reasonable amount for functional and structural studies. However, to optimize these systems without modifying the cellular functions of the living organism remains a challenging task. Cell-free expression systems have become a convenient method for the high-throughput expression of recombinant proteins, and great effort has been focused on generating high yields of proteins. Furthermore, these systems represent an attractive alternative for producing difficult-to-express proteins, such as membrane proteins. In this review, we highlight the recent improvements of these cell-free expression systems and their direct applications in the fields of membrane proteins production, protein therapy and modern proteomics.     

DNA甲基化与植物抗逆性研究进展

DNA甲基化是真核细胞基因组重要修饰方式之一.DNA甲基化通过与转录因子相互作用或通过改变染色质结构来影响基因的表达,从表观遗传水平对生物遗传信息进行调节,在生长发育过程中起着重要的作用,而且植物DNA甲基化还参与了环境胁迫下的基因表达调控过程.本文对植物DNA甲基化的产生机制、功能,以及DNA甲基化在植物应对逆境胁迫中的作用进行综述,以更好地理解植物DNA甲基化及其对环境胁迫的响应,为植物抗逆性研究及作物遗传改良提供理论参照.     

插入CpG序列的汉坦病毒S基因真核表达载体的构建及表达

To improve the effect of the gene immunization against Hantaan virus, we constructed the eukaryotic expression vector pTARGET-hans(ISS) containing Hantaan Virus S gene coding region and CpG motif by cloning S gene segment with CpG motif into eukaryotic expression vector pTARGET^TM.After conformed by enzyme analysis, the recombinant expression vector pTARGET-hans(ISS) was transferred into Vero-E6 cells by electroporation and the transient expression of Hantaan virus nucleocapsid protein was detected by indirect immunofluorescence assay(IFA). In some transferred Vero-E6, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector pTARGET-hans(ISS) was successfully constructed and expressed in vitro,which will lay a foundation for further animal vaccination.     

酵母作为外源基因表达系统的研究进展

甲醇营养型酵母和裂殖酵母作为外源基因表达的有效系统,正引起人们广泛研究。甲醇营养型酵母具有易诱导调控、适用于高密生长、能高效表达外源蛋白的特点。裂殖酵母有许多与高等真核细胞相似的特点,是研究真核分子生物学和真核基因表达有用的工具。本文综述了这两个酵母表达系统的特点。     

基因表达差异显示分析技术在创伤研究中的应用

基因是细胞增殖,分化,成熟等各项生命活动的调控中心,也是许多痢疾发生,发展和转归的决定性因素。基因表达的变化必然导致细胞,组织,器官乃至整个机体的各种异常。包括创伤在内的各种内外刺激,都可不同程度地引起基因表达的变化,最终妨碍机体健康。随着生物信息学的逐渐兴起和分子生物学的不断发展并向其他学科的逐渐渗透,业已建立起一系列研究基因表达变化的切实可行的技术手段（即“基因表达差异分析技术”,如DNA微阵列）,对捕获基因表达的种种变化具有重要价值。这些技术已经在肿瘤及其他疾病的研究中得到广泛应用,近几年也逐渐进入创伤研究领域,在一定程度上推动了创伤研究的发展。     

Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.     

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

We have engineered the chloroplast of eukaryotic algae to produce a number of recombinant proteins, including human monoclonal antibodies, but, to date, have achieved expression to only 0.5% of total protein. Here, we show that, by engineering the mammalian coding region of bovine mammary-associated serum amyloid (M-SAA) as a direct replacement for the chloroplast psbA coding region, we can achieve expression of recombinant protein above 5% of total protein. Chloroplast-expressed M-SAA accumulates predominantly as a soluble protein, contains the correct amino terminal sequence and has little or no post-translational modification. M-SAA is found in mammalian colostrum and stimulates the production of mucin in the gut, acting in the prophylaxis of bacterial and viral infections. Chloroplast-expressed and purified M-SAA is able to stimulate mucin production in human gut epithelial cell lines. As Chlamydomonas reinhardtii is an edible alga, production of therapeutic proteins in this organism offers the potential for oral delivery of gut-active proteins, such as M-SAA.     

Saccharomyces kluyveri as a model organism to study pyrimidine degradation

The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.     

The recombinant expression systems for structure determination of eukaryotic membrane proteins

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.